Consumption of peptide-included and free tryptophan induced by peroxyl radicals: A kinetic study.

PubMed

Fuentes, E; López-Alarcón, C

2014-10-01

It is well-known that tryptophan residues are efficiently oxidized by peroxyl radicals, generating kynurenine, and N-formyl kynurenine as well as hydroperoxide derivatives as products. In the present work we studied the kinetic of such reaction employing free and peptide-included tryptophan. Two azocompounds were used to produce peroxyl radicals: AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) and ABCVA (4,4'-Azobis(4-cyanovaleric acid)), which generate cationic and anionic peroxyl radicals, respectively. Tryptophan consumption was assessed by fluorescence spectroscopy and the reactions were carried out in phosphate buffer (75mM, pH 7.4) at 45°C. Only a slight effect of the peroxyl radical charge was evidenced on the consumption of free tryptophan and the dipeptide Gly-Trp. Employing AAPH as peroxyl radical source, at low free tryptophan concentrations (1-10µM) near 0.3 mol of tryptophan were consumed per each mol of peroxyl radicals introduced into the system. However, at high free tryptophan concentrations (100µM-1mM) such stoichiometry increased in a tryptophan concentration-way. At 1mM three moles of tryptophan were consumed per mol of AAPH-derived peroxyl radicals, evidencing the presence of chain reactions. A similar behavior was observed when di and tri-peptides (Gly-Trp, Trp-Gly, Gly-Trp-Gly, Trp-Ala, Ala-Trp-Ala) were studied. Nonetheless, at low initial concentration (5µM), the initial consumption rate of tryptophan included in the peptides was two times higher than free tryptophan. In contrast, at high concentration (1mM) free and peptide-included tryptophan showed similar initial consumption rates. These results could be explained considering a disproportionation process of tryptophanyl radicals at low free tryptophan concentrations, a process that would be inhibited when tryptophan is included in peptides. Copyright © 2014. Published by Elsevier Inc.

Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.

PubMed

Yang, Xiaolan; Hu, Xiaolei; Xu, Bangtian; Wang, Xin; Qin, Jialin; He, Chenxiong; Xie, Yanling; Li, Yuanli; Liu, Lin; Liao, Fei

2014-06-17

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

Fluorescence Determination of Tryptophan Side-Chain Accessibility and Dynamics in Triple-Helical Collagen-Like Peptides

PubMed Central

Simon-Lukasik, Kristine V.; Persikov, Anton V.; Brodsky, Barbara; Ramshaw, John A. M.; Laws, William R.; Alexander Ross, J. B.; Ludescher, Richard D.

2003-01-01

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)n. About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5–9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter (∼3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels. PMID:12524302

Changes in River Organic Matter Through Time.

NASA Astrophysics Data System (ADS)

Hudson, N.; Baker, A.; Ward, D.

2006-12-01

Samples of river water from central England were collected during the summer base-flow period. They were analysed for BOD and filtered at 1.2μm and 0.1μm increments to obtain i) the colloidal and dissolved, and ii) dissolved filter sterilized fractions. Each filtered fraction was plated up for microbiological cell counts and the agar plates and water samples were stored under a range of environmental conditions (4° C dark, 11° C light/ dark, 11° C dark, and 20° C dark) for 26 days. Absorbance, fluorescence, pH, conductivity and total organic carbon (TOC) were measured and colony forming units (CFU) counted on days 1, 2, 3, 4, 5, 12, 19 and 26. The fluorescence intensity was recorded for 5 commonly studied regions: protein like fluorescence, indicative of microbial activity, represented by the fluorescent amino acids tyrosine and tryptophan (which has two clear fluorescence regions) and humic and fulvic acids derived from the break down of terrestrial and aquatic plant material. Humic and fulvic-like fluorescence increased in all samples under all storage conditions suggesting that peaks A and C probably include a microbial element, either a product of the living community or as dead cell material in all fraction sizes including <0.1μm. Tryptophan and tyrosine-like fluorescence intensities demonstrated less clear trends which may be reflective of the intrinsic variation in natural samples. Tryptophan-like fluorescence generally decreased or showed minimal change, except in samples exposed to light in which an increase was observed in line with algal growth. A decrease in intensity may relate to the use of the tryptophan-like material as a microbial substrate. The increase in tryptophan-like fluorescence intensity suggests that this fluorescent material is being produced, either by algae, or bacterial activity associated with algal growth. It may also occur as a result of changing water chemistry causing a change in molecular conformation, and resulting fluorescence, as an increase in pH was also observed in these samples. This work illustrates the dynamic character of river organic matter within a timescale and under conditions that are representative of the natural system.

Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase Using Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro, C.; Gratson, A.A.; Evans, J.C.

2010-03-05

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind andmore » Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.« less

Distance Mapping in Proteins Using Fluorescence Spectroscopy: The Tryptophan-Induced Quenching (TrIQ) Method

PubMed Central

Mansoor, Steven E.; DeWitt, Mark A.; Farrens, David L.

2014-01-01

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such questions. This method, which we now call Tryptophan induced quenching (TrIQ), provides a straightforward, sensitive and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (~5 to 15 Å), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme and the extent of TrIQ caused by a nearby Trp was measured. Our results show that for smaller probes, TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, non-fluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or "sphere of quenching", which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based "sphere of quenching" is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. It provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy. PMID:20886836

Understanding aquatic microbial processes using EEM's and in-situ fluorescence sensors

NASA Astrophysics Data System (ADS)

Fox, Bethany; Attridge, John; Rushworth, Cathy; Cox, Tim; Anesio, Alexandre; Reynolds, Darren

2015-04-01

The diverse origin of dissolved organic matter (DOM) in aquatic systems is well documented within the literature. Previous literature indicates that coloured dissolved organic matter (CDOM) is, in part, transformed by aquatic microbial processes, and that dissolved organic material derived from a microbial origin exhibits tryptophan-like fluorescence. However, this phenomenon is not fully understood and very little data is available within the current literature. The overall aim of our work is to reveal the microbial-CDOM interactions that give rise to the observed tryptophan-like fluorescence. The work reported here investigates the microbial processes that occur within freshwater aquatic samples, as defined by the biochemical oxygen demand (BOD) test, as a function of the T1 peak (λex/em 280/330-370 nm). A series of standard water samples were prepared using glucose, glutamic acid, BOD dilution water and a bacterial seed (Cole-Parmer BOD microbe capsules). Samples were spiked with CDOM (derived from an environmental water body) and subjected to time resolved BOD analysis and as excitation-emission fluorescence spectroscopy. All EEM spectral data was interrogated using parallel factor analysis (PARAFAC) in an attempt to determine the presence and dominance (relative intensities) of the CDOM-related and T1-related fluorophores within the samples. In-situ fluorescence sensors (Chelsea Technologies Group Ltd.) were also used to monitor the T1 fluorescence peak (UviLux Tryptophan) and the CDOM fluorescence peak (UviLux CDOM) during experiments. Tryptophan-like fluorescence was observed (albeit transient) in both spiked and un-spiked standard water samples. By furthering our understanding of aquatic organic matter fluorescence, its origin, transformation, fate and interaction with aquatic microbiological processes, we aim to inform the design of a new generation in-situ fluorescence sensor for the monitoring of aquatic ecosystem health.

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

PubMed Central

Osterman, Ilya A.; Komarova, Ekaterina S.; Shiryaev, Dmitry I.; Korniltsev, Ilya A.; Khven, Irina M.; Lukyanov, Dmitry A.; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Efremenkova, Olga V.; Ivanenkov, Yan A.; Bogdanov, Alexey A.; Dontsova, Olga A.

2016-01-01

In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals. PMID:27736765

Enzyme-catalyzed Michael addition for the synthesis of warfarin and its determination via fluorescence quenching of L-tryptophan

NASA Astrophysics Data System (ADS)

Yuan, Yusheng; Yang, Liu; Liu, Shaopu; Yang, Jidong; Zhang, Hui; Yan, Jingjing; Hu, Xiaoli

2017-04-01

A sensitive fluorescence sensor for warfarin was proposed via quenching the fluorescence of L-tryptophan due to the interaction between warfarin and L-tryptophan. Warfarin, as one of the most effective anticoagulants, was designed and synthesized via lipase from porcine pancreas (PPL) as a biocatalyst to catalyze the Michael addition of 4-hydroxycoumarin to α, β-unsaturated enones in organic medium in the presence of water. Furthermore, the spectrofluorometry was used to detect the concentration of warfarin with a linear range and detection limit (3σ/k) of 0.04-12.0 μmol L- 1 (R2 = 0.994) and 0.01 μmol L- 1, respectively. Herein, this was the first application of bio-catalytic synthesis and fluorescence for the determination of warfarin. The proposed method was applied to determine warfarin of the drug in tablets with satisfactory results.

Tryptophan as key biomarker to detect gastrointestinal tract cancer using non-negative biochemical analysis of native fluorescence and Stokes Shift spectroscopy

NASA Astrophysics Data System (ADS)

Wang, Leana; Zhou, Yan; Liu, Cheng-hui; Zhou, Lixin; He, Yong; Pu, Yang; Nguyen, Thien An; Alfano, Robert R.

2015-03-01

The objective of this study was to find out the emission spectral fingerprints for discrimination of human colorectal and gastric cancer from normal tissue in vitro by applying native fluorescence. The native fluorescence (NFL) and Stokes shift spectra of seventy-two human cancerous and normal colorectal (colon, rectum) and gastric tissues were analyzed using three selected excitation wavelengths (e.g. 300 nm, 320 nm and 340 nm). Three distinct biomarkers, tryptophan, collagen and reduced nicotinamide adenine dinucleotide hydrate (NADH), were found in the samples of cancerous and normal tissues from eighteen subjects. The spectral profiles of tryptophan exhibited a sharp peak in cancerous colon tissues under a 300 nm excitation when compared with normal tissues. The changes in compositions of tryptophan, collagen, and NADH were found between colon cancer and normal tissues under an excitation of 300 nm by the non-negative basic biochemical component analysis (BBCA) model.

Characterization of interaction between amino acids and fulvic-like organic matter by fluorescence spectroscopy combining thermodynamic calculation.

PubMed

Lin, Tao; Hou, Bingwei; Wang, Jian; Xu, Yaqun; Chen, Wei

2017-03-01

Dissolved organic matter (DOM), as a very fine colloidal suspension, could inevitably affect the transformation process of dissolved organic nitrogen (DON) in drinking water treatment. Tryptophan and tyrosine were used as representatives of DON to investigate the interactions between amino acids and fulvic-like components of fluorescent DOM using titration experiments. The fluorescence intensity decreased significantly with the increasing fulvic acid (FA) concentration, suggesting that FA could greatly quench the intrinsic fluorescence of amino acids such as tryptophan and tyrosine. The absolute spectrum peaks of amino acids (AA) were changed in the presence of FA, possibly being resulted from non-covalent interactions between amino acids and FA. The specific hydrogen bonding and van der Waals forces played dominant roles in the interactions according to the results of theoretical analysis and thermodynamic calculation. The distance between donor and acceptor was 1.25 and 1.14 nm for the FA-tyrosine and FA-tryptophan system, indicating the energy transfer from tyrosine or tryptophan to FA. The association constant (K) decreased with the increase of temperature and pH value, while the change of ionic strength had no obvious influence on K value.

Alzheimer's disease evaluation using label-free, stainless, fluorescence to measure tryptophan metabolism along the kynurenine pathway

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Zhang, Lin; Shi, Lingyan; Sriramoju, Vidyasagar; Sordillo, Peter P.; Alfano, Robert R.

2018-02-01

Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer's disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer's disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer's patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer's and other neurodegenerative diseases.

A molecularly imprinted polymer-coated CdTe quantum dot nanocomposite for tryptophan recognition based on the Förster resonance energy transfer process

NASA Astrophysics Data System (ADS)

Tirado-Guizar, Antonio; Paraguay-Delgado, Francisco; Pina-Luis, Georgina E.

2016-12-01

A new ‘turn-on’ Förster resonance energy transfer (FRET) nanosensor for l-tryptophan based on molecularly imprinted quantum dots (QDs) is proposed. The approach combines the advantages of the molecular imprinting technique, the fluorescent characteristics of the QDs and the energy transfer process. Silica-coated CdTe QDs were first synthesized and then molecularly imprinted using a sol-gel process without surfactants. The final composite presents stable fluorescence which increases with the addition of l-tryptophan. This ‘turn-on’ response is due to a FRET mechanism from the l-tryptophan as donor to the imprinted QD as acceptor. QDs are rarely applied as acceptors in FRET systems. The nanosensor shows selectivity towards l-tryptophan in the presence of other amino acids and interfering ions. The l-tryptophan nanosensor exhibits a linear range between 0 and 8 µM concentration, a detection limit of 350 nM and high selectivity. The proposed sensor was successfully applied for the detection of l-tryptophan in saliva. This novel sensor may offer an alternative approach to the design of a new generation of imprinted nanomaterials for the recognition of different analytes.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

PubMed

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

PubMed Central

Gakamsky, Anna; Duncan, Rory R.; Howarth, Nicola M.; Dhillon, Baljean; Buttenschön, Kim K.; Daly, Daniel J.; Gakamsky, Dmitry

2017-01-01

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age. PMID:28071717

Correlation of tryptophan fluorescence intensity decay parameters with sup 1 H NMR-determined rotamer conformations: (tryptophan sup 2 )oxytocin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ross, J.B.A.; Schwartz, G.P.; Laws, W.R.

1992-02-18

While the fluorescence decay kinetics of tyrosine model compounds can be explained in terms of heterogeneity derived from the three ground-state {chi}{sup 1} rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from {chi}{sup 2} rotations. In this paper, the time-resolved and steady-state fluorescence properties of (tryptophan{sup 2})oxytocin at pH 3 are presented and compared with {sup 1}H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength-independent decay constants, only three exponential terms aremore » required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the {sup 1}H NMR-determined {chi}{sup 1} rotamer populations of the indole side chain. {sup 15}N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the {chi}{sup 1} rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two {chi}{sup 2} populations.« less

Potential rainfall-intensity and pH-driven shifts in the apparent fluorescent composition of dissolved organic matter in rainwater.

PubMed

Zhou, Yongqiang; Yao, Xiaolong; Zhang, Yibo; Shi, Kun; Zhang, Yunlin; Jeppesen, Erik; Gao, Guang; Zhu, Guangwei; Qin, Boqiang

2017-05-01

Perturbations of rainwater chromophoric dissolved organic matter (CDOM) fluorescence induced by changes in rainfall intensity and pH were investigated by field observations and laboratory pH titrations. Microbial humic-like fluorophores dominated the rainwater CDOM pool, followed by tryptophan-like and tyrosine-like substances. Increased rainfall intensity had notable dilution effects on all six fluorescent components (C1-C6) identified using parallel factor (PARAFAC) analysis, the effect being especially pronounced for the microbial humic-like C1, tryptophan-like C3, and tyrosine-like C5. The results also indicated that increasing pH from 7 to 9 led to decreased fluorescence intensity (F max ) of all the six components, while a pH increase from 5 to 7, resulted in increasing F max of terrestrial humic-like C2, tyrosine-like C5, and tryptophan-like C6 and decreasing microbial humic-like C1, tryptophan-like C3, and fulvic-like C4. Two-dimensional correlation spectroscopy (2D-COS) demonstrated that synchronous fluorescence responded first to pH modifications at fulvic-like wavelength (λ Ex /λ Em  = ∼316/416 nm), followed by tyrosine-like wavelength (λ Ex /λ Em  = ∼204/304 nm), tryptophan-like wavelength (λ Ex /λ Em  = ∼226/326 nm), microbial humic-like wavelength (∼295/395 nm), and finally terrestrial humic-like wavelength (∼360/460 nm). Our results suggest that a decrease in areas affected by acid rain in South China occurring at present may possibly result in apparent compositional changes of CDOM fluorescence. The decreased rainfall in South-West China and increased rainfall in North-West China during the past five decades may possibly accordingly result in increased and decreased F max of all the six components identified in South-West and North-West China, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fluorescence studies on native and bound to trifluraline soy bean Lb"a" in the enhanced N2 fixation.

PubMed

Kolev, K; Dolashka-Angelova, P

2001-10-01

The differences in the tryptophan (Trp) fluorescence of native (control) Lb"a" and experimental substance isolated from nodules of the Williams' soy beans variety treated with trifluraline at a concentration of 2.1 x 10(-10) M have been studied. A positively charged environment has been proved for the tryptophans of the native Lb"a" and a negative one for the tryptophans of the experimental Lb"a". The difference in the tryptophan emission spectra at lambdaex = 280 and 300 nm may be assigned to conformational alterations occurring in the experimental Lb"a". This is also confirmed by the greater energy transfer from tyrosine to tryptophan in the experimental Lb"a"--30% compared to the 10% in the native Lb"a". The value of the constant of acrylamide quenching (Ksv = 2.77 M(-1)) shows that the tryptophans are buried more deeply in the experimental Lb"a" than in the native Lb"a" (Ksv = 4 M(-1)). They are substantially lower than Ksv of the standard compound N-Ac-Trp-NH2 (16.30 M(-1)). The activation energy (Ea) of the thermal quenching of tryptophan fluorescence is higher for the experimental Lb"a" (37 kJ mol(-1)) as compared to the standard compound N-Ac-Trp-NH2 (24 kJ mol(-1)) and the native Lb "a" (32 kJ mol(-1)). The dissociation constant of the complex of trifluraline with Lb "a" (6.32 x 10(-11) M) has been determined as well as the stoichiometric ratio trifluraline/Lb"a" (1:1). The estimated nitrogenase activity (microM/gfrw h) and the total Lb (mg/gfrw) for trifluraline are higher as compared to those for the control.

Fluorescence studies on native and bound to trifluraline soy bean Lb "a" in the enhanced N 2 fixation

NASA Astrophysics Data System (ADS)

Kolev, Kolyo; Dolashka-Angelova, Pavlina

2001-10-01

The differences in the tryptophan (Trp) fluorescence of native (control) Lb "a" and experimental substance isolated from nodules of the 'Williams' soy beans variety treated with trifluraline at a concentration of 2.1×10 -10 M have been studied. A positively charged environment has been proved for the tryptophans of the native Lb "a" and a negative one for the tryptophans of the experimental Lb "a". The difference in the tryptophan emission spectra at λex=280 and 300 nm may be assigned to conformational alterations occurring in the experimental Lb "a". This is also confirmed by the greater energy transfer from tyrosine to tryptophan in the experimental Lb "a"—30% compared to the 10% in the native Lb "a". The value of the constant of acrylamide quenching ( Ksv=2.77 M -1) shows that the tryptophans are buried more deeply in the experimental Lb "a" than in the native Lb "a" ( Ksv=4 M -1). They are substantially lower than Ksv of the standard compound N-Ac-Trp-NH 2 (16.30 M -1). The activation energy ( Ea) of the thermal quenching of tryptophan fluorescence is higher for the experimental Lb "a" (37 kJ mol -1) as compared to the standard compound N-Ac-Trp-NH 2 (24 kJ mol -1) and the native Lb "a" (32 kJ mol -1). The dissociation constant of the complex of trifluraline with Lb "a" (6.32×10 -11 M) has been determined as well as the stoichiometric ratio trifluraline/Lb "a" (1:1). The estimated nitrogenase activity (μM/gfrw h) and the total Lb (mg/gfrw) for trifluraline are higher as compared to those for the control.

Intrinsic fluorescence biomarkers in cells treated with chemopreventive drugs

NASA Astrophysics Data System (ADS)

Kirkpatrick, Nathaniel D.; Brands, William R.; Zou, Changping; Brewer, Molly A.; Utzinger, Urs

2005-03-01

Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

PubMed

Li De La Sierra, I M; Vincent, M; Padron, G; Gallay, J

1992-01-01

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE PAGES

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.; ...

2016-05-19

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles.

PubMed

Lai, Chih Wei; Schwab, Mark; Hill, Steven C; Santarpia, Joshua; Pan, Yong-Le

2016-05-30

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300-350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm², dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ∼500-700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increased to 1 kW/cm² or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ∼ 650-800 nm with 633-nm CW laser excitation. We attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.

Correlation of conformational heterogeneity of the tryptophyl side chain and time-resolved fluorescence intensity decay kinetics

NASA Astrophysics Data System (ADS)

Laws, William R.; Ross, J. B. Alexander

1992-04-01

The time-resolved fluorescence properties of a tryptophan residue should be useful for probing protein structure, function, and dynamics. To date, however, the non-single exponential fluorescence intensity decay kinetics for numerous peptides and proteins having a single tryptophan residue have not been adequately explained. Many possibilities have been considered and include: (1) contributions from the 1La and 1Lb states of indole; (2) excited-state hydrogen exchange; and (3) environmental heterogeneity from (chi) 1 and (chi) 2 rotamers. In addition, it has been suggested that generally many factors contribute to the decay and a distribution of probabilities may be more appropriate. Two recent results support multiple species due to conformational heterogeneity as the major contributor to complex kinetics. First, a rotationally constrained tryptophan analogue has fluorescence intensity decay kinetics that can be described by the sum of two exponentials with amplitudes comparable to the relative populations of the two rotational isomers. Second, the multiple exponentials observed for tyrosine-containing model compounds and peptides correlate with the (chi) 1 rotamer populations independently determined by 1H NMR. We now report similar correlations between rotamer populations and fluorescence intensity decay kinetics for a tryptophan analogue of oxytocin. It appears for this compound that either (chi) 2 rotations do not appreciably alter the indole environment, (chi) 2 rotations are rapid enough to average the observed dependence, or only one of two possible (chi) 2 populations is associated with each (chi) 1 rotamer.

Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

PubMed Central

2015-01-01

Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context of spatially distinct differences in enthalpic barriers for protein conformational sampling that may be related to catalysis. PMID:24892947

Interaction of the major protein from bovine seminal plasma, PDC-109 with phospholipid membranes and soluble ligands investigated by fluorescence approaches.

PubMed

Anbazhagan, V; Damai, Rajani S; Paul, Aniruddha; Swamy, Musti J

2008-06-01

The major protein from bovine seminal plasma, PDC-109 binds selectively to choline phospholipids on the sperm plasma membrane and plays a crucial role in priming spermatozoa for fertilization. The microenvironment and accessibility of tryptophans of PDC-109 in the native state, in the presence of phosphorylcholine (PrC) and phospholipid membranes as well as upon denaturation have been investigated by fluorescence approaches. Quenching of the protein intrinsic fluorescence by different quenchers decreased in the order: acrylamide>succinimide>Cs(+)>I(-). Ligand binding afforded considerable protection from quenching, with shielding efficiencies following the order: dimyristoylphosphatidylcholine (DMPC)>lysophosphatidylcholine (Lyso-PC)>PrC. This has been attributed to a partial penetration of the protein into the DMPC membranes and Lyso-PC micelles, as well as a further stabilization of the binding due to the interaction of PDC-109 with lipid acyl chains and the resulting tightening of the protein structure, leading to a decreased accessibility of the tryptophan residues. Red-edge excitation shift (REES) studies yielded REES values of 4 nm for both native and denatured PDC-109, whereas reduced and denatured protein gave a REES of only 0.5 nm, clearly indicating that the structural and dynamic features of the microenvironment around the tryptophan residues are retained even after denaturation, presumably due to the constraints imposed on the protein structure by disulfide bonds. Upon binding of PDC-109 to DMPC membranes and Lyso-PC micelles the REES values were reduced to 2.5 and 1.0 nm, respectively, which could be due to the penetration of some parts of the protein, especially the segment containing Trp-90 into the membrane interior, where the red-edge effects are considerably reduced.

A Jerte Valley Cherry-Based Product as a Supply of Tryptophan

PubMed Central

Garrido, María; Espino, Javier; Toribio-Delgado, Antonio F.; Cubero, Javier; Maynar-Mariño, Juan I.; Barriga, Carmen; Paredes, Sergio D.; Rodríguez, Ana B.

2012-01-01

L-Tryptophan (tryptophan) is an essential amino acid in humans. It has important roles as a precursor of different bioactive compounds. Based on previous studies in which tryptophan has been shown to be present in fresh cherries, the aim of the present work was to analyze the tryptophan content of a Jerte Valley cherry-based product. A previously optimized method of analysis of tryptophan was used, ie, high-performance liquid chromatography with fluorescence detection (HPLC/FL). As expected, HPLC/FL technique permitted to detect and quantify the tryptophan content in a different matrix rather than fresh cherries. In fact, the Jerte Valley cherry-based product contained 69.54 ± 10.64 ppm of tryptophan, thereby showing that this product is a good source of tryptophan. In summary, it has been proven that the Jerte Valley cherry-based product is rich in tryptophan and may be indicated as a supply of this essential amino acid as well as having potential health benefits for conditions where tryptophan is necessary. PMID:22553424

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

PubMed

Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik

2017-06-01

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.

Interaction of amino acid-functionalized silver nanoparticles and Candida albicans polymorphs: A deep-UV fluorescence imaging study.

PubMed

Dojčilović, Radovan; Pajović, Jelena D; Božanić, Dušan K; Bogdanović, Una; Vodnik, Vesna V; Dimitrijević-Branković, Suzana; Miljković, Miona G; Kaščaková, Slavka; Réfrégiers, Matthieu; Djoković, Vladimir

2017-07-01

The interaction of the tryptophan functionalized Ag nanoparticles and live Candida albicans cells was studied by synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging at the DISCO beamline of Synchrotron SOLEIL. DUV imaging showed that incubation of the fungus with functionalized nanoparticles results in significant increase in the fluorescence signal. The analysis of the images revealed that the interaction of the nanoparticles with (pseudo)hyphae polymorphs of the diploid fungus was less pronounced than in the case of yeast cells or budding spores. The changes in the intensity of the fluorescence signals of the cells after incubation were followed in [327-353nm] and [370-410nm] spectral ranges that correspond to the fluorescence of tryptophan in non-polar and polar environment, respectively. As a consequence of the environmental sensitivity of the silver-tryptophan fluorescent nanoprobe, we were able to determine the possible accumulation sites of the nanoparticles. The analysis of the intensity decay kinetics showed that the photobleaching effects were more pronounced in the case of the functionalized nanoparticle treated cells. The results of time-integrated emission in the mentioned spectral ranges suggested that the nanoparticles penetrate the cells, but that the majority of the nanoparticles attach to the cells' surfaces. Copyright © 2017 Elsevier B.V. All rights reserved.

Self-assembled nanotubes from single fluorescent amino acid

NASA Astrophysics Data System (ADS)

Babar, Dipak Gorakh; Sarkar, Sabyasachi

2017-04-01

Self-assembly of biomolecules has gained increasing attention as it generates various supramolecular structural assemblies having potential applications principally in biomedical sciences. Here, we show that amino acid like tryptophan or tyrosine readily aggregates as nanotubes via a simple self-assembly process. These were characterized by FTIR, scanning electron microscopy, and by fluorescence microscopy. Nanotubes prepared from tryptophan are having 200 nm inner diameter and those from tyrosine are having the same around 50 nm diameter.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

PubMed

Huang, Cheng-Yen; Hsieh, Ming-Ching; Zhou, Qinwei

2017-04-01

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Tryptophan autofluorescence imaging of neoplasms of the human colon

NASA Astrophysics Data System (ADS)

Banerjee, Bhaskar; Renkoski, Timothy; Graves, Logan R.; Rial, Nathaniel S.; Tsikitis, Vassiliki Liana; Nfonsom, Valentine; Pugh, Judith; Tiwari, Piyush; Gavini, Hemanth; Utzinger, Urs

2012-01-01

Detection of flat neoplasia is a major challenge in colorectal cancer screening, as missed lesions can lead to the development of an unexpected `incident' cancer prior to the subsequent endoscopy. The use of a tryptophan-related autofluorescence has been reported to be increased in murine intestinal dysplasia. The emission spectra of cells isolated from human adenocarcinoma and normal mucosa of the colon were studied and showed markedly greater emission intensity from cancerous cells compared to cells obtained from the surrounding normal mucosa. A proto-type multispectral imaging system optimized for ultraviolet macroscopic imaging of tissue was used to obtain autofluorescence images of surgical specimens of colonic neoplasms and normal mucosa after resection. Fluorescence images did not display the expected greater emission from the tumor as compared to the normal mucosa, most probably due to increased optical absorption and scattering in the tumors. Increased fluorescence intensity in neoplasms was observed however, once fluorescence images were corrected using reflectance images. Tryptophan fluorescence alone may be useful in differentiating normal and cancerous cells, while in tissues its autofluorescence image divided by green reflectance may be useful in displaying neoplasms.

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

Real time monitoring of urban surface water quality using a submersible, tryptophan-like fluorescence sensor

NASA Astrophysics Data System (ADS)

Khamis, Kieran; Bradley, Chris; Hannah, David; Stevens, Rob

2014-05-01

Due to the recent development of field-deployable optical sensor technology, continuous quantification and characterization of surface water dissolved organic matter (DOM) is possible now. Tryptophan-like (T1) fluorescence has the potential to be a particularly useful indicator of human influence on water quality as T1 peaks are associated with the input of labial organic carbon (e.g. sewage or farm waste) and its microbial breakdown. Hence, real-time recording of T1 fluorescence could be particular useful for monitoring waste water infrastructure, treatment efficiency and the identification of contamination events at higher temporal resolution than available hitherto. However, an understanding of sensor measurement repeatability/transferability and interaction with environmental parameters (e.g. turbidity) is required. Here, to address this practical knowledge gap, we present results from a rigorous test of a commercially available submersible tryptophan fluorometer (λex 285, λem 350). Sensor performance was first examined in the laboratory by incrementally increasing turbidity under controlled conditions. Further to this the sensor was integrated into a multi-parameter sonde and field tests were undertaken involving: (i) a spatial sampling campaign across a range of surface water sites in the West Midlands, UK; and (ii) collection of high resolution (sub-hourly) samples from an urban stream (Bournbrook, Birmingham, U.K). To determine the ability of the sensor to capture spatiotemporal dynamics of urban waters DOM was characterized for each site or discrete time step using Excitation Emission Matrix spectroscopy and PARAFAC. In both field and laboratory settings fluorescence intensity was attenuated at high turbidity due to suspended particles increasing absorption and light scattering. For the spatial survey, instrument readings were compared to those obtained by a laboratory grade fluorometer (Varian Cary Eclipse) and a strong, linear relationship was apparent (R2 > 0.7). Parallel water sampling and laboratory analysis identified the potential for correction of T1 fluorescence intensity based on turbidity readings. These findings highlight the potential utility of real time monitoring of T1 fluorescence for a range of environmental applications (e.g. monitoring sewage treatment processes and tracing polluting DOM sources). However, if high/variable suspended sediment loads are anticipated concurrent monitoring of turbidity is required for accurate readings.

Conformational study of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) by tryptophan fluorescence and differential scanning calorimetry.

PubMed

Yin, Shou-Wei; Tang, Chuan-He; Yang, Xiao-Quan; Wen, Qi-Biao

2011-01-12

Fluorescence and differential scanning calorimetry (DSC) were used to study changes in the conformation of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) under various environmental conditions. The possible relationship between fluorescence data and DSC characteristics was also discussed. Tryptophan fluorescence and fluorescence quenching analyses indicated that the tryptophan residues in KPI, exhibiting multiple fluorophores with different accessibilities to acrylamide, are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains close to at least some of the tryptophan residues. GdnHCl was more effective than urea and SDS in denaturing KPI. SDS and urea caused variable red shifts, 2-5 nm, in the emission λ(max), suggesting the conformational compactness of KPI. The result was further supported by DSC characteristics that a discernible endothermic peak was still detected up to 8 M urea or 30 mM SDS, also evidenced by the absence of any shift in emission maximum (λ(max)) at different pH conditions. Marked decreases in T(d) and enthalpy (ΔH) were observed at extreme alkaline and/or acidic pH, whereas the presence of NaCl resulted in higher T(d) and ΔH, along with greater cooperativity of the transition. Decreases in T(d) and ΔH were observed in the presence of protein perturbants, for example, SDS and urea, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have a slight effect on the thermal properties of KPI. Interestingly, a close linear relationship between the T(d) (or ΔH) and the λ(max) was observed for KPI in the presence of 0-6 M urea.

Interaction of the iron(II) cage complexes with proteins: protein fluorescence quenching study.

PubMed

Losytskyy, Mykhaylo Y; Kovalska, Vladyslava B; Varzatskii, Oleg A; Sergeev, Alexander M; Yarmoluk, Sergiy M; Voloshin, Yan Z

2013-09-01

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.

Effect of disordered hemes and dimerization in isolated a-subunits of hemoglobin detected by time-resolved fluorescence spectroscopy in the picosecond range

NASA Astrophysics Data System (ADS)

Gryczynski, Zygmunt; Fronticelli, Clara; Gratton, Enrico; Lubkowski, Jacek; Bucci, Enrico

1994-08-01

Our recent linear dichroism study of transition moment directions for protoporphyrin derivatives [1,2] demonstrate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e. on the k2 orientation parameter of the Forster equation. Using the atomic coordinates of human hemoglobin and taking into account the direction of the transition moment of the near UV (300-380 nm) heme absorption band we have estimated the rate of energy transfer from tryptophan to heme in the isolated a chains, which are a single tryptophan protein. It appears that the rate of energy transfer is very sensitive to the orientation of the transition moment of the heme and similarly to myoglobin [3] natural heme disorder significantly reduces the transfer efficiency in isolated a subunits. On this basis we were able to predict very accurately the two lifetimes detectable in the systems, of 32 and 1050 ps respectively, where the amplitude of the longer lifetime is very consistent with the amount of disordered hemes found by La Mar [4,5] for the a subunits of hemoglobin.

Nonnegative matrix factorization: a blind sources separation method to extract content of fluorophores mixture media

NASA Astrophysics Data System (ADS)

Zhou, Kenneth J.; Chen, Jun

2014-03-01

The fluorophores of malignant human breast cells change their compositions that may be exposed in the fluorescence spectroscopy and blind source separation method. The content of the fluorophores mixture media such as tryptophan, collagen, elastin, NADH, and flavin were varied according to the cancer development. The native fluorescence spectra of these key fluorophores mixture media excited by the selective excitation wavelengths of 300 nm and 340 nm were analyzed using a blind source separation method: Nonnegative Matrix Factorization (NMF). The results show that the contribution from tryptophan, NADH and flavin to the fluorescence spectra of the mixture media is proportional to the content of each fluorophore. These data present a possibility that native fluorescence spectra decomposed by NMF can be used as potential native biomarkers for cancer detection evaluation of the cancer.

Spectroscopic properties and conformational stability of Concholepas concholepas hemocyanin.

PubMed

Idakieva, Krassimira; Nikolov, Peter; Chakarska, Irena; Genov, Nicolay; Shnyrov, Valery L

2008-01-01

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

NASA Astrophysics Data System (ADS)

Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

2013-06-01

A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

MiniFluo fluorescence sensor, advances in FDOM Ocean Measurements

NASA Astrophysics Data System (ADS)

Cyr, Frédéric; Tedetti, Marc; Goutx, Madeleine

2017-04-01

As part of the European project "Next generation Low-Cost Multifunctional Web Enabled Ocean Sensor Systems Empowering Marine, Maritime and Fisheries Management (NeXOS)", we developed the MiniFluo, a glider-compatible optical sensor for measurements of fluorescent dissolved organic matter (FDOM). In situ applications of the MiniFluo are presented here. The configuration used targets both natural (Tryptophan) and an anthropogenic (Phenanthrene) DOM fluorophores. Observations from three glider campaigns in the NW Mediterranean (Fall 2015 and Spring and Summer 2016) are presented. It is shown that the use of the Minifluo highlights new features of DOM dynamics in the region. For example, the Tryptophan (an amino-acid traditionally used as a tracer for waste waters) is found here closely related to open sea Chl-a fluorescence. Differences between Chl-a and Tryptophan fluorescence also give subtle information on seasonal changes in ecosystem structure and DOM release that could not be observed with traditional glider measurements. The study also highlights the presence of phenanthrene (an anthropogenic polycyclic aromatic hydrocarbon (PAH) in the surface and sub-surface waters of the Mediterranean. Implications of these finding will be put in the context of both the Mediterranean Sea DOM dynamics and also the ocean carbon cycle, from which the Dissolved Organic Carbon pool remains qualitatively unknown.

Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine

NASA Astrophysics Data System (ADS)

Shang, Xuefang; Li, Jie; Guo, Kerong; Ti, Tongyu; Wang, Tianyun; Zhang, Jinlian

2017-04-01

Inspired from biological counter parts, chemical modification of Schiff base derivatives with function groups may provide a highly efficient method to detect amino acids. Therefore, a fluorescent probe involving Schiff base and hydroxyl group has been designed and prepared, which showed high response and specificity for Arginine (Arg) among normal eighteen standard kinds of amino acids (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, the synthesized fluorescent probe exhibited high binding ability for Arg and low cytotoxicity to MCF-7 cells over a concentration range of 0-200 μg mL-1 which can be also used as a biosensor for the Arg detection in vivo.

Tryptophan and tryptophan-like substances in cloud water: Occurrence and photochemical fate

NASA Astrophysics Data System (ADS)

Bianco, Angelica; Passananti, Monica; Deguillaume, Laurent; Mailhot, Gilles; Brigante, Marcello

2016-07-01

This work investigates the occurrence and photochemical behaviour of tryptophan (TRP) in the cloud aqueous phase. The concentrations of tryptophan, TRYptophan LIke Substances (TRYLIS) and HUmic LIke Substances (HULIS) in real cloud water, collected between October 2013 and November 2014 at the top of the puy de Dôme station, were determined using the Excitation-Emission-Matrix (EEM) technique. The amount of free and complexed tryptophan (TRP) up to 10-7 M in cloud aqueous phase was quantified by HPLC-UV-fluorescence analysis, and its photoreactivity under sun-simulated conditions was investigated in synthetic water samples mimicking cloud aqueous phase compositions (oceanic and continental origins). TRP undergoes direct photolysis, and its degradation is enhanced in the presence of naturally occurring species able to photo-generate hydroxyl radicals (HOrad). The polychromatic quantum yield of TRP (ϕ290-340nmTRP) is estimated to be 8.37 × 10-4 between 290 and 340 nm, corresponding to the degradation rate (RTRPd) of 1.29 × 10-11 M s-1 under our irradiation conditions. The degradation is accelerated up to 3.65 × 10-10 and 8.26 × 10-10 M s-1 in synthetic oceanic and continental cloud water samples doped with 100 μM hydrogen peroxide, respectively. Hydroxyl radical-mediated transformation leads to the generation of different functionalized and oxidized products, as well as small carboxylic acids, such as formate and acetate. Moreover, fluorescent signals of irradiated solutions indicate the formation of HULIS.

Distance distributions of short polypeptides recovered by fluorescence resonance energy transfer in the 10 A domain.

PubMed

Sahoo, Harekrushna; Roccatano, Danilo; Zacharias, Martin; Nau, Werner M

2006-06-28

Fluorescence resonance energy transfer (FRET) between tryptophan (Trp) as donor and 2,3-diazabicyclo[2.2.2]oct-2-ene (Dbo) as acceptor was studied by steady-state and time-resolved fluorescence spectroscopy. The unique feature of this FRET pair is its exceptionally short Förster radius (10 A), which allows one to recover distance distributions in very short structureless peptides. The technique was applied to Trp-(GlySer)n-Dbo-NH2 peptides with n = 0-10, for which the average probe/quencher distance ranged between 8.7 and 13.7 A experimentally (in propylene glycol, analysis according to wormlike chain model) and 8.6-10.2 A theoretically (for n = 0-6, GROMOS96 molecular dynamics simulations). The larger FRET efficiency in steady-state compared to time-resolved fluorescence experiments was attributed to a static quenching component, suggesting that a small but significant part (ca. 10%) of the conformations are already in van der Waals contact when excitation occurs.

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

PubMed Central

Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

2016-01-01

ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread. PMID:27009950

Role of block copolymer-micelle nanocomposites in dye-bovine serum albumin binding: a combined experimental and molecular docking study.

PubMed

Manna, Anamika; Chakravorti, Sankar

2013-02-02

The role of a nanocomposite (NC), composed of intercalation of the diblock copolymer polyethylene-b-polyethylene glycol (PE-b-PEG) with the anionic surfactant sodium dodecyl sulphate (SDS), on the binding characteristics of bovine serum albumin (BSA) with a dye (1,8-naphthalimide, NAPMD) compared to the interaction between the same players in aqueous solution has been examined comprehensively in this paper. Static quenching due to complex formation in both NC medium and in buffer solution has been inferred on the basis of considerable changes in the absorption spectra of BSA on addition of NAPMD, of which the interaction is found to be stronger in NC medium. Temperature dependent fluorescence data also confirm an effective static quenching and stronger binding of NAPMD with BSA in NC medium. Peptide chain unfolding and denaturing of BSA in NC medium have been confirmed from steady state and time-resolved emission and circular dichroism data. This exposes both the tyrosine and tryptophan moieties as a unique case. Increased energy transfer between NAPMD and the tryptophan residue in the unfolded form of BSA helps in the appearance of tyrosine fluorescence in NC medium by quenching the tryptophan band. Ionization of the hydroxyl group in the aromatic ring of the tyrosine residue by the PEG group present in the NC medium produces a downshift of the tyrosine fluorescence band. The use of site selective markers confirms that NAPMD is near tryptophan in Sudlow's site I in NC medium and in buffer solution it is away from tryptophan in Sudlow's site II. The theoretical docking studies also vindicate the results of binding of NAPMD with BSA in site I or site II in NC and buffer media, as observed from different emission experiments including the site selective markers study.

Design and field application of a UV-LED based optical fiber biofilm sensor.

PubMed

Fischer, Matthias; Wahl, Martin; Friedrichs, Gernot

2012-03-15

Detecting changes in the formation dynamics of biofilms stemming from bacteria and unicellular microorganisms in their natural environment is of prime interest for biological, ecological as well as anti-fouling technology research. We developed a robust optical fiber-based biofilm sensor ready to be applied in natural aquatic environments for on-line, in situ and non-destructive monitoring of large-area biofilms. The device is based on the detection of the natural fluorescence of microorganisms constituting the biofilm. Basically, the intrinsic fluorescence of the amino acid tryptophan is excited at a wavelength of λ=280 nm and detected at λ=350 nm utilising a numerically optimized sensor head equipped with a UV-LED light source and optical fiber bundles for efficient fluorescence light collection. Calibration was carried out with tryptophan solutions and two characteristic marine bacteria strains revealing linear signal response, satisfactory background suppression, wide dynamic range, and an experimental detection limit of 4 × 10(3)cells/cm(2). Successful field experiments in the Baltic Sea accomplished over a period of twenty-one days provided for the first time continuous observation of biofilm formation dynamics in a natural habitat. Starting from the first adhering bacteria, the measurement yielded the characteristic three phases of biofilm formation up to a fully developed biofilm. The sensor system holds potential for applications in aquatic sciences including deep sea research and, after further miniaturisation, in the industrial and biomedical field. Copyright © 2012 Elsevier B.V. All rights reserved.

Quenching of Tryptophan Fluorescence in Unfolded Cytochrome "c": A Biophysics Experiment for Physical Chemistry Students

ERIC Educational Resources Information Center

Schlamadinger, Diana E.; Kats, Dina I.; Kim, Judy E.

2010-01-01

Laboratory experiments that focus on protein folding provide excellent opportunities for undergraduate students to learn important topics in the expanding interdisciplinary field of biophysics. Here, we describe the use of Stern-Volmer plots to determine the extent of solvent accessibility of the single tryptophan residue (trp-59) in unfolded and…

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

PubMed

Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-01-15

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

A comparison study of different excitation wavelengths to determine the relative content of key biomolecules in breast cancer and breast normal tissue

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, R. R.

2015-03-01

Fluorescence profiles from breast cancer and breast normal tissue samples with excitation wavelengths at 280 nm and 340 nm were obtained using the conventional LS-50 Perkin-Elmer spectrometer. Fluorescence ratios from these tissue samples, demonstrated by emission peaks at 340 nm, 440 nm and 460 nm and likely representing tryptophan and NADH, show increased relative content of tryptophan in malignant samples. Double ratio (DR) techniques were used to measure the severity of disease. The single excitation double ratio (Single-DR) method utilizes the emission intensity peaks from the spectrum acquired using a single excitation of 280 nm; while the dual excitation double ratio (dual-DR) method utilizes the emission intensity peaks from the spectra acquired using an excitation of 280 nm and 340 nm. Single-DR and dual-DR from 13 patients with breast carcinoma were compared in terms of their efficiency to distinguish high from low/intermediate tumors. Similar results were found with both methods. Results suggest that dual excitation wavelengths may be as effective as single excitation wavelength in calculating the relative content of biomolecules in breast cancer tissue, as well as for the assessment of the malignant potential of these tumors.

Assessing trihalomethanes (THMs) and N-nitrosodimethylamine (NDMA) formation potentials in drinking water treatment plants using fluorescence spectroscopy and parallel factor analysis.

PubMed

Yang, Liyang; Kim, Daekyun; Uzun, Habibullah; Karanfil, Tanju; Hur, Jin

2015-02-01

The formation of disinfection byproducts (DBPs) is a major challenge in drinking water treatments. This study explored the applicability of fluorescence excitation-emission matrices and parallel factor analysis (EEM-PARAFAC) for assessing the formation potentials (FPs) of trihalomethanes (THMs) and N-nitrosodimethylamine (NDMA), and the treatability of THM and NDMA precursors in nine drinking water treatment plants. Two humic-like and one tryptophan-like components were identified for the samples using PARAFAC. The total THM FP (TTHM FP) correlated strongly with humic-like component C2 (r=0.874), while NDMA FP showed a moderate and significant correlation with the tryptophan-like component C3 (r=0.628). The reduction by conventional treatment was more effective for C2 than C3, and for TTHM FP than NDMA FP. The treatability of DOM and TTHM FP correlated negatively with the absorption spectral slope (S275-295) and biological index (BIX) of the raw water, but it correlated positively with humification index (HIX). Our results demonstrated that PARAFAC components were valuable for assessing DBPs FP in drinking water treatments, and also that the raw water quality could affect the treatment efficiency. Copyright © 2014 Elsevier Ltd. All rights reserved.

Water Quality Monitoring Using Tryptophan-like Fluorescence.

NASA Astrophysics Data System (ADS)

Hudson, N.; Urquhart, G.; Baker, A.; Ward, D.; Reynolds, D.; Carliell-Marquet, C.

2006-12-01

The Biochemical Oxygen Demand (BOD) test is recognised as being credible with over 90 years of application in water analysis. However it is easily affected by environmental constraints and requires the presence of a viable biological community. The BOD test takes 5-21 days and is laboratory based and so is unsuitable for rapid responses to potential pollution incidents. Analysis of fluorescence excitation emission matrices (EEM) of natural waters gives a rapid determination of the proportions of labile and refractory organic matter present. It facilitates a greater understanding of the oxygen depleting potential of organic matter in unfiltered samples in a shorter timescale than would be the case using BOD, the conventional water quality assessment method. The research presented assesses the relationship between 5-day BOD (BOD5) and the fluorescent amino acid tryptophan-like peak for a range of waters. The research is undertaken with a view to using fluorescence spectroscopy as an alternative to the BOD5 test for on-site monitoring or lab based, rapid indication of organic pollution in natural waters. A significant relationship is observed between the analytical parameters in line with the findings of previous research in which waste waters including synthetic sewage, and polluted surface waters were studied. This research demonstrates that for a large, variable data set tryptophan-like fluorescence is a strong indicator of BOD5 and may be used as a water quality monitoring tool particularly for high BOD5 samples.

Deciphering the fluorescence resonance energy transfer from denatured transport protein to anthracene 1,5 disulphonate in reverse micellar environment

NASA Astrophysics Data System (ADS)

Singharoy, Dipti; Bhattacharya, Subhash Chandra

2017-12-01

Constrained environmental effect inside AOT reverse micellar media has been employed in this work to collect the information about energy transfer efficacy between sodium salt of anthracene 1,5 disulphonate (1,5-AS) with model transport proteins, bovine serum albumin (BSA), and human serum albumin (HSA). Steady state, time-resolved fluorescence and circular dichroism techniques have been used for this purpose and corresponding Fӧrster-type resonance energy transfer (FRET) from tryptophan residues to 1,5-AS indicates that 1,5-AS binds in the vicinity of the tryptophan residue (BSA and HSA) with equal strength. Indication of protein damage from fluorescence data and its confirmation has been measured from CD measurement. Molecular modeling study hereby plays a crucial role to predict the minimum energy docked conformation of the probe inside the protein environment. From the docked conformation the distance between 1,5-AS and tryptophan moiety of BSA/HSA has successfully explained the FRET possibility between them. A comparative modeling study between BSA and HSA with 1,5-AS assigning their binding site within specific amino acids plays a crucial role in support of the FRET study.

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model.

PubMed

Wang, Ying; Gutierrez-Herrera, Enoch; Ortega-Martinez, Antonio; Anderson, Richard Rox; Franco, Walfre

2016-09-01

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross-links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model. Non-closing non-grafted, partial closing non-grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide-field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization. The endogenous fluorescence excitation of cross-links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non-closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial-close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement. We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin-digestible collagen cross-links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678-685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Fluorescence calibration method for single-particle aerosol fluorescence instruments

NASA Astrophysics Data System (ADS)

Shipley Robinson, Ellis; Gao, Ru-Shan; Schwarz, Joshua P.; Fahey, David W.; Perring, Anne E.

2017-05-01

Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310-400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420-650 nm). The relationship between fluorescence and mass for the mixed tryptophan-ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hill, Steven C.; Williamson, Chatt C.; Doughty, David C.

This paper uses a mathematical model of fluorescent biological particles composed of bacteria and/or proteins (mostly as in Hill et al., 2013 [23]) to investigate the size-dependence of the total fluorescence emitted in all directions. The model applies to particles which have negligible reabsorption of fluorescence within the particle. The specific particles modeled here are composed of ovalbumin and of a generic Bacillus. The particles need not be spherical, and in some cases need not be homogeneous. However, the results calculated in this paper are for spherical homogeneous particles. Light absorbing and fluorescing molecules included in the model are aminomore » acids, nucleic acids, and several coenzymes. Here the excitation wavelength is 266 nm. The emission range, 300 to 370 nm, encompasses the fluorescence of tryptophan. The fluorescence cross section (C F) is calculated and compared with one set of published measured values. We investigate power law (Ad y) approximations to C F, where d is diameter, and A and y are parameters adjusted to fit the data, and examine how y varies with d and composition, including the fraction as water. The particle's fluorescence efficiency (Q F=C F/geometric-cross-section) can be written for homogeneous particles as Q absR F, where Q abs is the absorption efficiency, and R F, the fraction of the absorbed light emitted as fluorescence, is independent of size and shape. When Q F is plotted vs. m id or mi(m r-1)d, where m=m r+im i is the complex refractive index, the plots for different fractions of water in the particle tend to overlap.« less

Size-dependent fluorescence of bioaerosols: Mathematical model using fluorescing and absorbing molecules in bacteria

DOE PAGES

Hill, Steven C.; Williamson, Chatt C.; Doughty, David C.; ...

2015-02-02

This paper uses a mathematical model of fluorescent biological particles composed of bacteria and/or proteins (mostly as in Hill et al., 2013 [23]) to investigate the size-dependence of the total fluorescence emitted in all directions. The model applies to particles which have negligible reabsorption of fluorescence within the particle. The specific particles modeled here are composed of ovalbumin and of a generic Bacillus. The particles need not be spherical, and in some cases need not be homogeneous. However, the results calculated in this paper are for spherical homogeneous particles. Light absorbing and fluorescing molecules included in the model are aminomore » acids, nucleic acids, and several coenzymes. Here the excitation wavelength is 266 nm. The emission range, 300 to 370 nm, encompasses the fluorescence of tryptophan. The fluorescence cross section (C F) is calculated and compared with one set of published measured values. We investigate power law (Ad y) approximations to C F, where d is diameter, and A and y are parameters adjusted to fit the data, and examine how y varies with d and composition, including the fraction as water. The particle's fluorescence efficiency (Q F=C F/geometric-cross-section) can be written for homogeneous particles as Q absR F, where Q abs is the absorption efficiency, and R F, the fraction of the absorbed light emitted as fluorescence, is independent of size and shape. When Q F is plotted vs. m id or mi(m r-1)d, where m=m r+im i is the complex refractive index, the plots for different fractions of water in the particle tend to overlap.« less

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Two-channel dansyl/tryptophan emitters with a cholic acid bridge as reporters for local hydrophobicity within supramolecular systems based on bile salts.

PubMed

Gomez-Mendoza, M; Marin, M Luisa; Miranda, Miguel A

2014-11-14

The aim of the present work is to develop two-channel emitters to probe local hydrophobicity by means of fluorescence quenching within different biomimetic supramolecular environments. To achieve this goal, the dansyl (Dns) and tryptophan (Trp) fluorophores have been covalently attached to cholic acid (CA) in order to ensure simultaneous incorporation of the two emitting units into the same compartment. In principle, the two fluorophores of the synthesized Dns-CA-Trp probes could either exhibit an orthogonal behavior or display excited state interactions. The fluorescence spectra of 3β-Dns-CA-Trp showed a residual Trp emission band at ca. 350 nm and an enhanced Dns maximum in the 500-550 nm region. This reveals a partial intramolecular energy transfer, which is consistent with the Dns and Trp singlet energies. Thus, the two photoactive units are not orthogonal; nevertheless, 3β-Dns-CA-Trp seems appropriate as a two-channel reporter for the supramolecular systems of interest. Fluorescence quenching of 3β-Dns-CA-Trp by iodide (which remains essentially in bulk water) was examined within sodium cholate, sodium taurocholate, sodium deoxycholate and mixed micelles. Interestingly, a decrease in the emission intensity of the two bands was observed with increasing iodide concentrations. The most remarkable effect was observed for mixed micelles, where the quenching rate constants were one order of magnitude lower than in solution. As anticipated, the quenching efficiency by iodide decreased with increasing hydrophobicity of the microenvironment, a trend that can be correlated with the relative accessibility of the probe to the ionic quencher.

Nonnegative constraint analysis of key fluorophores within human breast cancer using native fluorescence spectroscopy excited by selective wavelength of 300 nm

NASA Astrophysics Data System (ADS)

Pu, Yang; Sordillo, Laura A.; Alfano, Robert R.

2015-03-01

Native fluorescence spectroscopy offers an important role in cancer discrimination. It is widely acknowledged that the emission spectrum of tissue is a superposition of spectra of various salient fluorophores. In this study, the native fluorescence spectra of human cancerous and normal breast tissues excited by selected wavelength of 300 nm are used to investigate the key building block fluorophores: tryptophan and reduced nicotinamide adenine dinucleotide (NADH). The basis spectra of these key fluorophores' contribution to the tissue emission spectra are obtained by nonnegative constraint analysis. The emission spectra of human cancerous and normal tissue samples are projected onto the fluorophore spectral subspace. Since previous studies indicate that tryptophan and NADH are key fluorophores related with tumor evolution, it is essential to obtain their information from tissue fluorescence but discard the redundancy. To evaluate the efficacy of for cancer detection, linear discriminant analysis (LDA) classifier is used to evaluate the sensitivity, and specificity. This research demonstrates that the native fluorescence spectroscopy measurements are effective to detect changes of fluorophores' compositions in tissues due to the development of cancer.

Utilization of fluorescence spectroscopy as a novel approach to evaluate the oxidative stability in beef retail displayed.

PubMed

Pouzo, L B; Zaritzky, N E; Pavan, E; Rossetti, L; Descalzo, A M

2016-09-01

Beef samples from grazing steers finished with different seed-supplemented diets were vacuum packaged for 3, 14 and 56days (VC) and subsequently exposed to aerobic conditions (AE) for 0 and 5days. Different fluorescent compounds of interest in the oxidation process were detected in meat, namely tryptophan residues, Schiff bases and porphyrins. Tryptophan intensity fluorescence increased with 14days of VC; while Schiff bases intensity increased (P<0.05) in beef samples stored under VC-56 and in all samples after AE-5days. Porphyrins increased (P<0.05) gradually with the extension of vacuum storage time, but were degraded in beef with long vacuum storage and 5days of AE. Higher levels of porphyrins in beef under VC were correlated (P<0.05) with lower redness and higher TBARS after AE-5. This study revealed the potential of fluorescence signals to detect oxidative changes in beef under different storage conditions using a fast and nondestructive method such as fluorescence spectroscopy. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Broken Ring: Reduced Aromaticity in Lys-Trp Cations and High pH Tautomer Correlates with Lower Quantum Yield and Shorter Lifetimes

PubMed Central

2015-01-01

Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK1, pK2, and pKR) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the 1La transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes. PMID:24882092

Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies

NASA Astrophysics Data System (ADS)

Maiti, Jyotirmay; Biswas, Suman; Chaudhuri, Ankur; Chakraborty, Sandipan; Chakraborty, Sibani; Das, Ranjan

2017-03-01

An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift ( 10 nm) and smaller Stokes' shift ( 5980 cm- 1) in BSA than HSA (Stokes'shift 6600 cm- 1), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (Ka 5.2 × 106 M- 1) than the DMOBA-HSA complex (Ka 1.0 × 106 M- 1). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5 Å) than HSA (25.4 Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the formation of the HSA-DMOBA complex. Electrostatic interactions contribute significantly in the binding of DMOBA to HSA (- 2.09 kcal/mol) compared to BSA (- 0.47 kcal/mol). Electrostatic surface potential calculation reveals that the DMOBA binding site within HSA is highly charged compared to BSA.

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

PubMed

Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M

2015-01-01

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.

High histologic grade and increased relative content of tryptophan in breast cancer using ratios from fingerprint fluorescence spectral peaks

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, R. R.

2015-03-01

Histologic grade is a very important, but underappreciated, parameter of breast cancer aggressiveness. Despite its importance, it has historically not been included as one of the criteria for staging of this cancer. In this study, spectral fluorescence profiles from patients with breast carcinoma were acquired. Ratios of emission peaks at 340 over 440,460 nm from biomolecules in malignant and normal samples were calculated. Cancerous over normal ratios (double ratio (DR) method) were evaluated with respect to tumor characteristics. Increased tryptophan content in breast cancer tissues correlates strongly with high grade, but not with lymph node metastases, estrogen receptor, progesterone receptor or Her-2-Neu receptor status.

Time-resolved fluorescence of thioredoxin single-tryptophan mutants: modeling experimental results with minimum perturbation mapping

NASA Astrophysics Data System (ADS)

Silva, Norberto D., Jr.; Haydock, Christopher; Prendergast, Franklyn G.

1994-08-01

The time-resolved fluorescence decay of single tryptophan (Trp) proteins is typically described using either a distribution of lifetimes or a sum of two or more exponential terms. A possible interpretation for this fluorescence decay heterogeneity is the existence of different isomeric conformations of Trp about its (chi) +1) and (chi) +2) dihedral angles. Are multiple Trp conformations compatible with the remainder of the protein in its crystallographic configuration or do they require repacking of neighbor side chains? It is conceivable that isomers of the neighbor side chains interconvert slowly on the fluorescence timescale and contribute additional lifetime components to the fluorescence intensity. We have explored this possibility by performing minimum perturbation mapping simulations of Trp 28 and Trp 31 in thioredoxin (TRX) using CHARMm 22. Mappings of Trp 29 and Trp 31 give the TRX Trp residue energy landscape as a function of (chi) +1) and (chi) +2) dihedral angles. Time-resolved fluorescence intensity and anisotropy decay of mutant TRX (W28F and W31F) are measured and interpreted in light of the above simulations. Relevant observables, like order parameters and isomerization rates, can be derived from the minimum perturbation maps and compared with experiment.

Fluorescence spectra of blood plasma treated with ultraviolet irradiation in vivo

NASA Astrophysics Data System (ADS)

Zalesskaya, G. A.; Maslova, T. O.

2010-09-01

We have studied the fluorescence spectra of blood plasma from patients with acute coronary syndrome, and also the effect of therapeutic doses of in vivo ultraviolet blood irradiation (UBI) on the spectra. We have established that the maxima in the fluorescence spectra of the original plasma samples, obtained from unirradiated blood, are located in the wavelength interval 330-340 nm, characteristic for the fluorescence of tryptophan residues. In extracorporeal UBI ( λ = 254 nm), we observed changes in the shape and also both a blue and a red shift in the maxima of the fluorescence spectra, differing in magnitude for blood plasma samples from different patients in the test group. We show that UBI-initiated changes in the fluorescence spectra of the plasma depend on the original pathological disturbances of metabolite levels, and also on the change in the oxygen-transport function of the blood and the acid-base balance, affecting the oxidative stability of the plasma. We have concluded that UV irradiation, activating buffer systems in the blood, has an effect on the universal and specific interactions of the tryptophan residue with the amino acid residues and water surrounding it.

Concentration, size, and excitation power effects on fluorescence from microdroplets and microparticles containing tryptophan and bacteria

NASA Astrophysics Data System (ADS)

Fell, Nicholas F., Jr.; Pinnick, Ronald G.; Hill, Steven C.; Videen, Gorden W.; Niles, Stanley; Chang, Richard K.; Holler, Stephen; Pan, Yongle; Bottiger, Jerold R.; Bronk, Burt V.

1999-01-01

Our group has been developing a system for single-particle fluorescence detection of aerosolized agents. This paper describes the most recent steps in the evolution of this system. The effects of fluorophore concentrations, droplet size, and excitation power have also been investigated with microdroplets containing tryptophan in water to determine the effects of these parameters on our previous results. The vibrating orifice droplet generator was chosen for this study base don its ability to generate particles of well- known and reproducible size. The power levels required to reach saturation and photodegradation were determined. In addition, the collection of fluorescence emission was optimized through the use of a UV achromatic photographic lens. This arrangement permitted collection of images of the droplet stream. Finally, the use of a dual-beam, conditional firing scheme facilitated the collection of improved signal- to-noise single-shot spectra from individual biological particles.

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

NASA Astrophysics Data System (ADS)

Nemtseva, Elena V.; Lashchuk, Olesya O.; Gerasimova, Marina A.; Melnik, Tatiana N.; Nagibina, Galina S.; Melnik, Bogdan S.

2018-01-01

In most cases, intermediate states of multistage folding proteins are not ‘visible’ under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

PubMed

Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

2017-12-21

In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

Comprehensive spectroscopic studies on the interaction of biomolecules with surfactant detached multi-walled carbon nanotubes.

PubMed

Sekar, Gajalakshmi; Mukherjee, Amitava; Chandrasekaran, Natarajan

2015-04-01

This paper investigates the interaction of ten diverse biomolecules with surfactant detached Multi-Walled Carbon Nanotubes (MWCNTs) using multiple spectroscopic methods. Declining fluorescence intensity of biomolecules in combination with the hyperchromic effect in UV-Visible spectra confirmed the existence of the ground state complex formation. Quenching mechanism remains static and non-fluorescent. 3D spectral data of biomolecules suggested the possibilities of disturbances to the aromatic microenvironment of tryptophan and tyrosine residues arising out of CNTs interaction. Amide band Shifts corresponding to the secondary structure of biomolecules were observed in the of FTIR and FT-Raman spectra. In addition, there exists an increased Raman intensity of tryptophan residues of biomolecules upon interaction with CNTs. Hence, the binding of the aromatic structures of CNTs with the aromatic amino acid residues, in a particular, tryptophan was evidenced. Far UV Circular spectra have showed the loss of alpha-helical contents in biomolecules upon interaction with CNTs. Near UV CD spectra confirmed the alterations in the tryptophan positions of the peptide backbone. Hence, our results have demonstrated that the interaction of biomolecules with OH-MWCNTs would involve binding cum structural changes and alteration to their aromatic micro-environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Short communication: Suitability of fluorescence spectroscopy for characterization of commercial milk of different composition and origin.

PubMed

Ntakatsane, M P; Yang, X Q; Lin, M; Liu, X M; Zhou, P

2011-11-01

Thirteen milk brands comprising 76 pasteurized and UHT milk samples of various compositions (whole, reduced fat, skimmed, low lactose, and high protein) were obtained from local supermarkets, and milk samples manufactured in various countries were discriminated using front-face fluorescence spectroscopy (FFFS) coupled with chemometric tools. The emission spectra of Maillard reaction products and riboflavin (MRP/RF; 400 to 600 nm) and tryptophan (300 to 400 nm) were recorded using FFFS, and the excitation wavelengths were set at 360 nm for MRP/RF and 290 nm for tryptophan. Principal component analysis (PCA) was applied to analyze the normalized spectra. The PCA of spectral information from MRP/RF discriminated the milk samples originating in different countries, and PCA of spectral information from tryptophan discriminated the samples according to composition. The fluorescence spectral data were compared with liquid chromatography-mass spectrometry results for the glycation extent of the milk samples, and a positive association (R(2)=0.84) was found between the degree of glycation of α-lactalbumin and the MRP/RF spectral data. This study demonstrates the ability and sensitivity of FFFS to rapidly discriminate and classify commercial milk with various compositions and processing conditions. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

PubMed

Rawel, Harshadrai M; Frey, Simone K; Meidtner, Karina; Kroll, Jürgen; Schweigert, Florian J

2006-08-01

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Inhibition of Tryptophan on AA 2024 in Chloride-Containing Solutions

NASA Astrophysics Data System (ADS)

Li, Xing; Xiang, Bin; Zuo, Xiu-Li; Wang, Qin; Wei, Zi-Dong

2011-03-01

The inhibitory effects of tryptophan on the corrosion of AA 2024 in 1 M HCl, 20% (wt.%) CaCl2, and 3.5% (wt.%) NaCl solutions were investigated via polarization techniques, electrochemical impedance spectroscopy, and weight loss methods. The scanning electron microscope technique was employed to observe corrosion morphology. The results suggest that AA 2024 was corroded in these three corrosive media to some extent and that tryptophan can significantly inhibit the corrosion of aluminum alloys. The inhibition efficiency (η) increased with increasing concentrations of tryptophan, and the best inhibition efficiency exhibited was about 87% in 1 M HCl solution with 0.008 M tryptophan. Tryptophan acted as a cathodic corrosion inhibitor and affected the hydrogen evolution reaction, which was the main electrode reaction in the 1 M HCl solution. In solutions with 20% CaCl2 and 3.5% NaCl, tryptophan was adsorbed onto anodic areas, thus increasing the activation energy of the interface reaction as an anodic corrosion inhibitor. The Dmol3 program of Material Studio 4.0 was used to obtain the optimized geometry of the tryptophan inhibitor and some quantum-chemical parameters. Front orbital distributions and Fukui indices indicate that the molecular active reaction zones were located in the indole ring of tryptophan.

Spectroscopie pompe-sonde pour la détection de bioaérosols

NASA Astrophysics Data System (ADS)

Guyon, L.; Courvoisier, F.; Wood, V.; Boutou, V.; Bartelt, A.; Roth, M.; Rabitz, H.; Wolf, J. P.

2006-10-01

La fluorescence du Tryptophane excité par une impulsion ultra-brève à 270 nm peut être diminuée d'un facteur deux par une seconde impulsion à 800 nm, à l'aide d'un dispositif pompe-sonde. Cette décroissance est aussi observée pour les bactéries vivantes, dont le Tryptophane est l'un des fluorophores, tandis qu'aucune décroissance n'est observée pour d'autres molécules organiques comme le naphtalène ou le gazole, malgré des spectres d'absorption et de fluorescence similaires. Cette différence remarquable est très prometteuse pour la distinction d'aérosols biologiques et organiques.

Real-time detection of faecally contaminated drinking water with tryptophan-like fluorescence: defining threshold values.

PubMed

Sorensen, James P R; Baker, Andy; Cumberland, Susan A; Lapworth, Dan J; MacDonald, Alan M; Pedley, Steve; Taylor, Richard G; Ward, Jade S T

2018-05-01

We assess the use of fluorescent dissolved organic matter at excitation-emission wavelengths of 280nm and 360nm, termed tryptophan-like fluorescence (TLF), as an indicator of faecally contaminated drinking water. A significant logistic regression model was developed using TLF as a predictor of thermotolerant coliforms (TTCs) using data from groundwater- and surface water-derived drinking water sources in India, Malawi, South Africa and Zambia. A TLF threshold of 1.3ppb dissolved tryptophan was selected to classify TTC contamination. Validation of the TLF threshold indicated a false-negative error rate of 15% and a false-positive error rate of 18%. The threshold was unsuccessful at classifying contaminated sources containing <10 TTC cfu per 100mL, which we consider the current limit of detection. If only sources above this limit were classified, the false-negative error rate was very low at 4%. TLF intensity was very strongly correlated with TTC concentration (ρ s =0.80). A higher threshold of 6.9ppb dissolved tryptophan is proposed to indicate heavily contaminated sources (≥100 TTC cfu per 100mL). Current commercially available fluorimeters are easy-to-use, suitable for use online and in remote environments, require neither reagents nor consumables, and crucially provide an instantaneous reading. TLF measurements are not appreciably impaired by common intereferents, such as pH, turbidity and temperature, within typical natural ranges. The technology is a viable option for the real-time screening of faecally contaminated drinking water globally. Copyright © 2017 Natural Environment Research Council (NERC), as represented by the British Geological Survey (BGS. Published by Elsevier B.V. All rights reserved.

Greatly enhanced binding of a cationic porphyrin towards bovine serum albumin by cucurbit[8]uril.

PubMed

Lei, Wanhua; Jiang, Guoyu; Zhou, Qianxiong; Zhang, Baowen; Wang, Xuesong

2010-10-28

Binding affinity towards serum albumin and intracellular proteins is of importance for a photodynamic therapy (PDT) sensitizer to selectively localize in tumours and efficiently induce cell death. In this paper, it was found that cucurbit[8]uril (CB8) can greatly improve the binding affinity of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), a promising PDT photosensitizer, towards bovine serum albumin (BSA). Absorption, fluorescence emission, (1)H NMR, dynamic light scattering, atomic force microscope, as well as protein photocleavage measurements suggest that the binding enhancement originates from the formation of a ternary complex of CB8·TMPyP·tryptophan residues. This finding opens up a new approach for the development of more efficient PDT agents.

Room temperature fluorescence and phosphorescence study on the interactions of iodide ions with single tryptophan containing serum albumins

NASA Astrophysics Data System (ADS)

Gałęcki, Krystian; Kowalska-Baron, Agnieszka

2016-12-01

In this study, the influence of heavy-atom perturbation, induced by the addition of iodide ions, on the fluorescence and phosphorescence decay parameters of some single tryptophan containing serum albumins isolated from: human (HSA), equine (ESA) and leporine (LSA) has been studied. The obtained results indicated that, there exist two distinct conformations of the proteins with different exposure to the quencher. In addition, the Stern-Volmer plots indicated saturation of iodide ions in the binding region. Therefore, to determine quenching parameter, we proposed alternative quenching model and we have performed a global analysis of each conformer to define the effect of iodide ions in the cavity by determining the value of the association constant. The possible quenching mechanism may be based on long-range through-space interactions between the buried chromophore and quencher in the aqueous phase. The discrepancies of the decay parameters between the albumins studied may be related with the accumulation of positive charge at the main and the back entrance to the Drug Site 1 where tryptophan residue is located.

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

NASA Astrophysics Data System (ADS)

Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

2016-09-01

The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496-502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

Parametric models to compute tryptophan fluorescence wavelengths from classical protein simulations.

PubMed

Lopez, Alvaro J; Martínez, Leandro

2018-02-26

Fluorescence spectroscopy is an important method to study protein conformational dynamics and solvation structures. Tryptophan (Trp) residues are the most important and practical intrinsic probes for protein fluorescence due to the variability of their fluorescence wavelengths: Trp residues emit in wavelengths ranging from 308 to 360 nm depending on the local molecular environment. Fluorescence involves electronic transitions, thus its computational modeling is a challenging task. We show that it is possible to predict the wavelength of emission of a Trp residue from classical molecular dynamics simulations by computing the solvent-accessible surface area or the electrostatic interaction between the indole group and the rest of the system. Linear parametric models are obtained to predict the maximum emission wavelengths with standard errors of the order 5 nm. In a set of 19 proteins with emission wavelengths ranging from 308 to 352 nm, the best model predicts the maximum wavelength of emission with a standard error of 4.89 nm and a quadratic Pearson correlation coefficient of 0.81. These models can be used for the interpretation of fluorescence spectra of proteins with multiple Trp residues, or for which local Trp environmental variability exists and can be probed by classical molecular dynamics simulations. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

PubMed

Iijima, Issei; Hohsaka, Takahiro

2009-04-17

Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

PubMed Central

Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation. PMID:26656259

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability.

PubMed

Oliveira Silva, Catarina; Petersen, Steffen B; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.

Fluorescence quenching effects of antibiotics on the main components of dissolved organic matter.

PubMed

Yan, Peng-Fei; Hu, Zhen-Hu; Yu, Han-Qing; Li, Wei-Hua; Liu, Li

2016-03-01

Dissolved organic matter (DOM) in wastewater can be characterized using fluorescence excitation-emission matrix and parallel factor (EEM-PARAFAC) analysis. Wastewater from animal farms or pharmaceutical plants usually contains high concentration of antibiotics. In this study, the quenching effect of antibiotics on the typical components of DOM was explored using fluorescence EEM-PARAFAC analysis. Four antibiotics (roxarsone, sulfaquinoxaline sodium, oxytetracycline, and erythromycin) at the concentration of 0.5∼4.0 mg/L and three typical components of DOM (tyrosine, tryptophan, and humic acid) were selected. Fluorescence quenching effects were observed with the addition of antibiotics. Among these four antibiotics, roxarsone (2.9∼20.2 %), sulfaquinoxaline sodium (0∼32.0 %), and oxytetracycline (0∼41.8 %) led to a stronger quenching effect than erythromycin (0∼8.0 %). From the side of DOM, tyrosine and tryptophan (0.5∼41.8 %) exhibited a similar quenching effect, but they were higher than humic acids (0∼20.2 %) at the same concentration of antibiotics. For humic acid, a significant quenching effect was observed only with the addition of roxarsone. This might be the first report about the fluorescence quenching effect caused by antibiotics. The results from this study confirmed the interference of antibiotics on the fluorescence intensity of the main components of DOM and highlighted the importance of correcting fluorescence data in the wastewater containing antibiotics.

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

PubMed Central

Feinstein, Efraim; Deikus, Gintaras; Rusinova, Elena; Rachofsky, Edward L.; Ross, J. B. Alexander; Laws, William R.

2003-01-01

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay. PMID:12524313

Multi-spectroscopic investigation on the complexation of tetracycline with dissolved organic matter derived from algae and macrophyte.

PubMed

Bai, Leilei; Zhao, Zhen; Wang, Chunliu; Wang, Changhui; Liu, Xin; Jiang, Helong

2017-11-01

Interactions of antibiotics with algae-derived dissolved organic matter (ADOM) and macrophyte-derived dissolved organic matter (MDOM) are of vital importance to the transport and ecotoxicity of antibiotics in eutrophic freshwater lakes. Multi-spectroscopic techniques were used to investigate the complexation of tetracycline (TTC) with ADOM and MDOM collected from Lake Taihu (China). The 3 fluorescent components, tyrosine-, tryptophan-, and humic-like component, were identified by excitation emission matrix spectra with parallel factor analysis. Their fluorescence was quenched at different degree by TTC titration through static quenching. The complexation of TTC induced conformational changes in DOM fractions. Synchronous fluorescence spectra combined with two dimensional correlation spectroscopy further suggested that the formation of TTC-DOM complexes occurred on the sequential order of tryptophan-like→tyrosine-like→humic-like component. The effective quenching constants of tryptophan- and tyrosine-like component were similar, higher than those of humic-like component. The strong binding ability and abundant content of protein-like substances indicated their prominent role in the TTC-DOM complexation. Fourier transform infrared spectroscopy further revealed that the heterogeneous functional groups, including amide I and II, aromatics, and aliphatics, were responsible for the complexation. These results highlight the significant impact of the overgrowth of algae and macrophyte on the environmental behavior of antibiotics in waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

The damaging effect of UV-C irradiation on lens alpha-crystallin.

PubMed

Fujii, Noriko; Uchida, Hiroki; Saito, Takeshi

2004-11-02

To evaluate the effect of UV-C irradiation on the structural properties of alpha-crystallin and its chaperone activity. alpha- and betaL-crystallins were isolated from bovine lenses using gel chromatography. The purified alpha-crystallin was subjected to UV-C irradiation (254 nm; 1, 2, 5, 10, 20, 50 J/cm2). We measured the tryptophan fluorescence, circular dichroism (CD) spectroscopy in the far UV, and the chaperone activity of both irradiated and non-irradiated alpha-crystallin. The tryptophan fluorescence of alpha-crystallin decreased, whereas the N-formylkynurenine fluorescence increased markedly with increasing doses of UV-C irradiation. Both the oxidation of Met1 and the racemization of Asp151 of alphaA-crystallin increased at a dose of 1-2 J/cm2 and then gradually decreased. The CD spectrum showed that the secondary structure of alpha-crystallin altered with increasing radiation dose, and almost all of the beta-sheet structure was lost at doses above 50 J/cm2. The chaperone activity of alpha-crystallin irradiated with doses under 5 J/cm2 remained intact. However, it was reduced to only 40% after irradiation at 10 J/cm2. Our study suggests that photo-oxidation of tryptophan residues in alpha-crystallin may be one of the events that affects the three-dimensional packing array and chaperone activity of this lens protein.

Determination of Time since Death using Vitreous Humor Tryptophan.

PubMed

Ansari, Niha; Menon, Shobhana K

2017-09-01

Determination of time since death (TSD) plays very important role in forensic examination as it narrows down field of suspects and aids in deceased identification. This study utilizes the fluorescence property of vitreous humor (VH) tryptophan to determine TSD using o-phthalaldehyde (OPA). The detection limit of these fluorometric studies was found to be 8 ppb indicating sensitivity and high accuracy in TSD determination. The study was performed on selected 76 cadaver with known TSD ranging from 3 to 90 h. Excellent correlation between VH tryptophan and TSD was obtained with a coefficient of correlation R 2 = 0.9590. Results showed statistically significant increase in vitreous tryptophan with TSD up to 90 h, and the proposed method was efficaciously applied for prediction of TSD as no systematic error exist. The regression equation obtained from the study is [Trp] = 2.21 + 2.98 * TSD. © 2017 American Academy of Forensic Sciences.

Calcium-Induced Conformational Transition of Trout Ependymins Monitored by Tryptophan Fluorescence

PubMed Central

Ganss, Bernhard; Hoffmann, Werner

2009-01-01

Ependymins are secretory, calcium-binding sialoproteins which are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these regeneration-responsive glycoproteins is associated with collagen fibrils of the extracellular matrix. Here, the tryptophan fluorescence of ependymins was monitored at various Ca2+ concentrations. Two distinct states were identified with a relatively sharp transition at about 1 mM Ca2+. In agreement with previous circular dichroism measurements, this strongly supports the hypothesis that a calcium-induced conformational change is important for the interaction of ependymins with components of the extracellular matrix. Such interactions with constituents of various basal laminae would also explain the important roles of piscine ependymins as well as invertebrate and mammalian ependymin-related proteins for cell adhesion processes and cell migration. PMID:19401757

Calcium-induced conformational transition of trout ependymins monitored by tryptophan fluorescence.

PubMed

Ganss, Bernhard; Hoffmann, Werner

2009-01-01

Ependymins are secretory, calcium-binding sialoproteins which are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these regeneration-responsive glycoproteins is associated with collagen fibrils of the extracellular matrix. Here, the tryptophan fluorescence of ependymins was monitored at various Ca(2+) concentrations. Two distinct states were identified with a relatively sharp transition at about 1 mM Ca(2+). In agreement with previous circular dichroism measurements, this strongly supports the hypothesis that a calcium-induced conformational change is important for the interaction of ependymins with components of the extracellular matrix. Such interactions with constituents of various basal laminae would also explain the important roles of piscine ependymins as well as invertebrate and mammalian ependymin-related proteins for cell adhesion processes and cell migration.

Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, P.K.; Lee, Cheng S.; King, J.A.

1997-12-31

The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresismore » (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.« less

The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

NASA Astrophysics Data System (ADS)

Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

2016-03-01

The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

Novel cookie-with-chocolate carbon dots displaying extremely acidophilic high luminescence

NASA Astrophysics Data System (ADS)

Lu, Siyu; Zhao, Xiaohuan; Zhu, Shoujun; Song, Yubin; Yang, Bai

2014-10-01

A fluorescent carbon dot with a cookie-with-chocolate film structure (about 5 × 5 μm2) showed a high fluorescence quantum yield (61.12%) at low pH. It was hydrothermally synthesized from l-serine and l-tryptophan. The formation mechanism of the film with carbon dots (CDs) was investigated. The film structure was formed by hydrogen bonding and π-π stacking interactions between aromatic rings. The strong blue fluorescence of the CDs increased under strong acidic conditions owing to the changes in the N-groups. These cookie-like CDs are attractive for their potential use as effective fluorescent probes for the sensitive detection of aqueous H+ and Fe3+.A fluorescent carbon dot with a cookie-with-chocolate film structure (about 5 × 5 μm2) showed a high fluorescence quantum yield (61.12%) at low pH. It was hydrothermally synthesized from l-serine and l-tryptophan. The formation mechanism of the film with carbon dots (CDs) was investigated. The film structure was formed by hydrogen bonding and π-π stacking interactions between aromatic rings. The strong blue fluorescence of the CDs increased under strong acidic conditions owing to the changes in the N-groups. These cookie-like CDs are attractive for their potential use as effective fluorescent probes for the sensitive detection of aqueous H+ and Fe3+. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03965c

Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies.

PubMed

Maiti, Jyotirmay; Biswas, Suman; Chaudhuri, Ankur; Chakraborty, Sandipan; Chakraborty, Sibani; Das, Ranjan

2017-03-15

An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift (~10nm) and smaller Stokes' shift (~5980cm -1 ) in BSA than HSA (Stokes'shift~6600cm -1 ), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (K a ~5.2×10 6 M -1 ) than the DMOBA-HSA complex (K a ~1.0×10 6 M -1 ). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5Å) than HSA (25.4Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the formation of the HSA-DMOBA complex. Electrostatic interactions contribute significantly in the binding of DMOBA to HSA (-2.09kcal/mol) compared to BSA (-0.47kcal/mol). Electrostatic surface potential calculation reveals that the DMOBA binding site within HSA is highly charged compared to BSA. Copyright © 2016 Elsevier B.V. All rights reserved.

Accumulation of humic-like and proteinaceous dissolved organic matter in zero-discharge aquaculture systems as revealed by fluorescence EEM spectroscopy.

PubMed

Yamin, G; Borisover, M; Cohen, E; van Rijn, J

2017-01-01

Recirculating aquaculture systems (RAS), offering many economic and fish husbandry benefits, are characterized by an accumulation of dissolved organic matter (DOM) and, specifically, humic substances (HS). As reported in a number of studies, HS may affect biological activity in both invertebrates and vertebrates. Given the accumulation of HS in RAS, it is therefore of great interest to characterize DOM and, specifically, its HS fraction in the RAS. The present study was aimed at characterizing long-term changes in fluorescent DOM composition in the culture water of RAS systems, which were operated in a novel, zero water exchange mode. Two such zero-discharge recirculating systems (ZDS) were examined: a freshwater system, stocked with hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and a marine system, stocked with gilthead seabream (Sparus aurata). Excitation-emission matrices (EEMs) of fluorescence, coupled with parallel factor analysis (PARAFAC), were used to characterize and quantify the different DOM components in the ZDS. In the culture water, one tryptophan-like and four HS-like components were identified. The fluorescence intensities of three of the HS-like components as well as the tryptophan-like component increased at comparable rates during ZDS operation while a much slower accumulation of these compounds was observed in a parallel operated, flow-through, freshwater aquarium. The ZDS examined in this study comprised a sludge digestion stage where a considerable accumulation of all fluorescent components was detected. A HS-like components and a tryptophan-like component in blood of tilapia from the freshwater ZDS were similar to components found in the culture water. Blood levels of both components were higher in fish cultured in the DOM-rich ZDS than in fish raised in the control, flow-through freshwater aquarium. Fluorescence of the HS-like component found in the fish blood increased also with time of ZDS operation. The finding that fish blood contains a HS-like fluorescent component may have important implications for the understanding of the physiological effects of HS in fish and the possible benefits of these substances in aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differentiation of cancerous and normal brain tissue using label free fluorescence and Stokes shift spectroscopy

NASA Astrophysics Data System (ADS)

Zhou, Yan; Wang, Leana; Liu, Cheng-hui; He, Yong; Yu, Xinguang; Cheng, Gangge; Wang, Peng; Shu, Cheng; Alfano, Robert R.

2016-03-01

In this report, optical biopsy was applied to diagnose human brain cancer in vitro for the identification of brain cancer from normal tissues by native fluorescence and Stokes shift spectra (SSS). 77 brain specimens including three types of human brain tissues (normal, glioma and brain metastasis of lung cancers) were studied. In order to observe spectral changes of fluorophores via fluorescence, the selected excitation wavelength of UV at 300 and 340 nm for emission spectra and a different Stokes Shift spectra with intervals Δλ = 40 nm were measured. The fluorescence spectra and SSS from multiple key native molecular markers, such as tryptophan, collagen, NADH, alanine, ceroid and lipofuscin were observed in normal and diseased brain tissues. Two diagnostic criteria were established based on the ratios of the peak intensities and peak position in both fluorescence and SSS spectra. It was observed that the ratio of the spectral peak intensity of tryptophan (340 nm) to NADH (440 nm) increased in glioma, meningioma (benign), malignant meninges tumor, and brain metastasis of lung cancer tissues in comparison with normal tissues. The ratio of the SS spectral peak (Δλ = 40 nm) intensities from 292 nm to 366 nm had risen similarly in all grades of tumors.

Tracking variations in fluorescent-dissolved organic matter in an aerobic submerged membrane bioreactor using excitation-emission matrix spectra combined with parallel factor analysis.

PubMed

Hur, Jin; Shin, Jaewon; Kang, Minsun; Cho, Jinwoo

2014-08-01

In this study, the variations in the fluorescent components of dissolved organic matter (DOM) were tracked for an aerobic submerged membrane bioreactor (MBR) at three different operation stages (cake layer formation, condensation, and after cleaning). The fluorescent DOM was characterized using excitation-emission matrix (EEM) spectroscopy combined with parallel factor analysis (PARAFAC). Non-aromatic carbon structures appear to be actively involved in the membrane fouling for the cake layer formation stage as revealed by much higher UV-absorbing DOM per organic carbon found in the effluent versus those inside the reactor. Four fluorescent components were successfully identified from the reactor and the effluent DOMs by EEM-PARAFAC modeling. Among those in the reactor, microbial humic-like fluorescence was the most abundant component at the cake layer formation stage and tryptophan-like fluorescence at the condensation stage. In contrast to the reactor, relatively similar composition of the PARAFAC components was exhibited for the effluent at all three stages. Tryptophan-like fluorescence displayed the largest difference between the reactor and the effluent, suggesting that this component could be a good tracer for membrane fouling. It appears that the fluorescent DOM was involved in membrane fouling by cake layer formation rather than by internal pore adsorption because its difference between the reactor and the effluent was the highest among all the four components, even after the membrane cleaning. Our study provided an insight into the fate and the behavior fluorescent DOM components for an MBR system, which could be an indicator of the membrane fouling.

Ultrafast fluorescence spectroscopy via upconversion applications to biophysics.

PubMed

Xu, Jianhua; Knutson, Jay R

2008-01-01

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.

Hand-held synchronous scan spectrometer for in situ and immediate detection of live/dead bacteria ratio

NASA Astrophysics Data System (ADS)

Li, Runze; Goswami, Umang; Walck, Matthew; Khan, Kasfia; Chen, Jie; Cesario, Thomas C.; Rentzepis, Peter M.

2017-11-01

The design, construction, and operation of a hand-held synchronously scanned, excitation-emission, double monochromator spectrometer is described. Data show that it is possible to record and display within minutes the fluorescence spectra and ratio of live/dead bacteria in situ. Excitation emission matrix contour plots display clearly bacteria fluorescence spectra before and after UV inactivation, respectively. The separation of the fluorescence band maxima of molecular components, such as tryptophan, tyrosine, and DNA, may be distinguished in the diffused fluorescence spectra of bacteria and mixtures.

Hand-held synchronous scan spectrometer for in situ and immediate detection of live/dead bacteria ratio.

PubMed

Li, Runze; Goswami, Umang; Walck, Matthew; Khan, Kasfia; Chen, Jie; Cesario, Thomas C; Rentzepis, Peter M

2017-11-01

The design, construction, and operation of a hand-held synchronously scanned, excitation-emission, double monochromator spectrometer is described. Data show that it is possible to record and display within minutes the fluorescence spectra and ratio of live/dead bacteria in situ. Excitation emission matrix contour plots display clearly bacteria fluorescence spectra before and after UV inactivation, respectively. The separation of the fluorescence band maxima of molecular components, such as tryptophan, tyrosine, and DNA, may be distinguished in the diffused fluorescence spectra of bacteria and mixtures.

Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods

PubMed Central

Chaari, Ali; Fahy, Christine; Chevillot-Biraud, Alexandre; Rholam, Mohamed

2015-01-01

Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer’s and Parkinson’s diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils. PMID:26571264

Thermostability of glucose oxidase in silica gel obtained by sol-gel method and in solution studied by fluorimetric method.

PubMed

Przybyt, Małgorzata; Miller, Ewa; Szreder, Tomasz

2011-04-04

The thermostability of glucose oxidase entrapped in silica gel obtained by sol-gel method was studied by thermostimulated fluorescence of FAD at pH 5 and 7 and compared with that of the native enzyme in the solution and at the presence of ethanol. The unfolding temperatures were found to be lower for the enzyme immobilised in gel as compared with the native enzyme but higher as for the enzyme at the presence of ethanol. In gel, the thermal denaturation of glucose oxidase is independent on pH while in solution the enzyme is more stable at pH 5. The investigation the enzyme in different environment by steady-state fluorescence of FAD and tryptophan, synchronous fluorescence and time-resolved fluorescence of tryptophan indicates that the state of the molecule (tertiary structure and molecular dynamics) is different in gel and in solution. The ethanol produced during gel precursor hydrolysis is not the main factor influencing the thermostability of the enzyme but more important are interactions of the protein with the gel lattice. Copyright © 2011 Elsevier B.V. All rights reserved.

Tryptophan fluorescence in the Bacillus subtilis phototropin-related protein YtvA as a marker of interdomain interaction.

PubMed

Losi, Aba; Ternelli, Elena; Gärtner, Wolfgang

2004-01-01

The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N-terminal light, oxygen and voltage (LOV) domain. The blue light-triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot-LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA-LOV are markedly different from those observed in the full-length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA-LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN-cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light-regulated energy transfer process from a yet unidentified tyrosine to W103.

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching*

PubMed Central

Billen, Bert; Brams, Marijke; Debaveye, Sarah; Remeeva, Alina; Alpizar, Yeranddy A.; Waelkens, Etienne; Kreir, Mohamed; Brüggemann, Andrea; Talavera, Karel; Nilius, Bernd; Voets, Thomas; Ulens, Chris

2015-01-01

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. PMID:25829496

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics

PubMed Central

Xu, Jianhua; Knutson, Jay R.

2012-01-01

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed. PMID:19152860

Effects of environmental lighting and tryptophan devoid diet on the rat vaginal cycle.

PubMed

Giammanco, S; Ernandes, M; La Guardia, M

1997-09-01

Cerebral serotonin level influences luteinizing hormone release and, consequently, ovulation. The present study evaluated the effects of precooked maize meal (polenta), a diet almost devoid of tryptophan the serotonin precursor on the alterations of the estrus cycle as measured by vaginal smears analysis in Wistar rats. Several conditions of environmental lighting were used in order to modify ovarian cycle: 1) natural alternating light/dark cycle; 2) continuous darkness; 3) continuous light by sodium steams: 4) continuous light by fluorescent neon tubes. Rats bred in continuous lighting showed estrus-proestrus rate significantly greater than rats bred in normal lighting or in continuous darkness. The feeding with precooked maize meal suppressed persistent estrus in rats bred in continuous lighting, and significantly cut down the estrus-proestrus frequency in any condition of environmental lighting. Our results lead to hypothesize that polenta diet, for its low tryptophan content, cutting down both tryptophan plasma content and serotonin neuronal synthesis, promotes luteinizing hormone peak.

Synchrotron Ultraviolet Microspectroscopy on Rat Cortical Bone: Involvement of Tyrosine and Tryptophan in the Osteocyte and Its Environment

PubMed Central

Pallu, Stéphane; Rochefort, Gael Y.; Jaffre, Christelle; Refregiers, Matthieu; Maurel, Delphine B.; Benaitreau, Delphine; Lespessailles, Eric; Jamme, Frédéric; Chappard, Christine; Benhamou, Claude-Laurent

2012-01-01

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately. PMID:22937127

Lactoferrin denaturation induced by anionic surfactants: The role of the ferric ion in the protein stabilization.

PubMed

Ferreira, Gabriel Max Dias; Ferreira, Guilherme Max Dias; Agudelo, Álvaro Javier Patiño; Hudson, Eliara Acipreste; Dos Santos Pires, Ana Clarissa; da Silva, Luis Henrique Mendes

2018-05-11

Here, investigation was made of the interaction between Lactoferrin (Lf) and the anionic surfactants sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), and sodium decyl sulfate (DSS), using isothermal titration calorimetry, Nano differential scanning calorimetry (NanoDSC), and fluorescence spectroscopy. The Lf-surfactant interaction was enthalpically favorable (the integral enthalpy change ranged from -5.99 kJ mol -1 , for SDS at pH 3.0, to -0.61 kJ mol -1 , for DSS at pH 12.0) and promoted denaturation of the protein. The Lf denaturation efficiency followed the order DSS < SDS < SDBS. The extent of binding of the surfactants to Lf strongly depended on pH and the surfactant structure, reaching a maximum value of 505 SDBS molecules per gram of Lf at pH 3.0. The different efficiencies of the surfactants in denaturing Lf were attributed to the balance of hydrophobic and electrostatic interactions, which also depended on pH and the surfactant structure, highlighting the SDBS-tryptophan residue specific interaction, where SDBS acted as a quencher of fluorescence. Interestingly, the NanoDSC and fluorescence measurements showed that the ferric ion bound to Lf increased its stability against denaturation induced by the surfactants. The results have important implications for understanding the influence of surfactants on structural changes in metalloproteins. Copyright © 2017. Published by Elsevier B.V.

Applying a mathematical model to estimate the fractional accessibility to quenching of serum albumin by risperidone

NASA Astrophysics Data System (ADS)

Carqueja, Marilena; Cortez, Celia Martins

2014-10-01

In this work we report the results from application of a mathematical model to estimate the fractional accessibility to fluorescence quenching by risperidone in human and bovine sera albumins. Risperidone is an atypical antipsychotic drug used to treat many kinds of psychiatric disorders. Results showed that but the fractional accessibility for trypyophan 134, sub domain 1B, is about 3 times higher than that to tryptophan 212, showing that the primary binding site for risperidone is close to tryptophan 134, in domain IB of BSA.

Crystal structure of phototoxic orange fluorescent proteins with α tryptophan-based chromophore

DOE PAGES

Pletneva, Nadya V.; Pletnev, Vladimir Z.; Sarkisyan, Karen S.; ...

2015-12-23

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. We present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKiller-Orange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enablingmore » transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.« less

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

PubMed

Buijs; Hlady

1997-06-01

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

PubMed

Abe, Ryoji; Ohashi, Hiroyuki; Iijima, Issei; Ihara, Masaki; Takagi, Hiroaki; Hohsaka, Takahiro; Ueda, Hiroshi

2011-11-02

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore

PubMed Central

Pletneva, Nadya V.; Pletnev, Vladimir Z.; Sarkisyan, Karen S.; Gorbachev, Dmitry A.; Egorov, Evgeny S.; Mishin, Alexander S.; Lukyanov, Konstantin A.; Dauter, Zbigniew; Pletnev, Sergei

2015-01-01

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers. PMID:26699366

A fluorescence approach to the unfolding thermodynamics of horseradish peroxidase based on heme degradation by hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ke, Zhigang; Ma, Shanshan; Li, Lamei; Huang, Qing

2016-07-01

Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H2O2) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods.

Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-02-16

Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold

NASA Astrophysics Data System (ADS)

Cheng, Zihao; Campbell, Robert E.

2007-02-01

Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.

FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

NASA Astrophysics Data System (ADS)

O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Rehman, Shagufta; Periasamy, Ammasi

2016-03-01

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes -- rather than whole cell averages - makes this assay particularly valuable.

Study on interaction between a new fluorescent probe 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one and BSA.

PubMed

Qiu, Bin; Guo, Longhua; Chen, Mingluan; Lin, Zhenyu; Chen, Guonan

2011-03-07

A new fluorescence reagent, 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one (mBPO), synthesized in our laboratory was used as the probe for protein and its interaction with Bovine Serum Albumin (BSA) was investigated in detail in this paper. It was found that BSA had the ability to quench the fluorescence of mBPO at 411 nm (λ(ex) = 286 nm), and the quenched intensity of fluorescence was proportional to the concentration of BSA. Based on this fact, mBPO has been used as a fluorescence probe for the detection of BSA. Under the optimal conditions, the calibration graph is linear up to 0.5 mg L(-1) for BSA and the limit of detection (LOD) was 0.06 mg L(-1). The regression equation is y = 1048.8x + 7.2093 with R(2) = 0.9913. The mechanism for the interaction of mBPO with BSA was also studied, while the binding constant and the number of binding sites were calculated. According to the thermodynamics parameter, the binding mode between mBPO and BSA was deduced. The results suggested the interaction between mBPO and BSA to be hydrophobic force in nature. It also proved that the fluorescence quenching reaction was affected by the tryptophan residue of BSA. For there are two tryptophan (Trp) residues, in site 134 and site 212 of BSA, and mBPO maybe has interaction with them respectively.

PARAFAC Modeling of Irradiation- and Oxidation-Induced Changes in Fluorescent Dissolved Organic Matter Extracted from Poultry Litter.

PubMed

Mangalgiri, Kiranmayi P; Timko, Stephen A; Gonsior, Michael; Blaney, Lee

2017-07-18

Parallel factor analysis (PARAFAC) applied to fluorescence excitation emission matrices (EEMs) allows quantitative assessment of the composition of fluorescent dissolved organic matter (DOM). In this study, we fit a four-component EEM-PARAFAC model to characterize DOM extracted from poultry litter. The data set included fluorescence EEMs from 291 untreated, irradiated (253.7 nm, 310-410 nm), and oxidized (UV-H 2 O 2 , ozone) poultry litter extracts. The four components were identified as microbial humic-, terrestrial humic-, tyrosine-, and tryptophan-like fluorescent signatures. The Tucker's congruence coefficients for components from the global (i.e., aggregated sample set) model and local (i.e., single poultry litter source) models were greater than 0.99, suggesting that the global EEM-PARAFAC model may be suitable to study poultry litter DOM from individual sources. In general, the transformation trends of the four fluorescence components were comparable for all poultry litter sources tested. For irradiation at 253.7 nm, ozonation, and UV-H 2 O 2 advanced oxidation, transformation of the humic-like components was slower than that of the tryptophan-like component. The opposite trend was observed for irradiation at 310-410 nm, due to differences in UV absorbance properties of components. Compared to the other EEM-PARAFAC components, the tyrosine-like component was fairly recalcitrant in irradiation and oxidation processes. This novel application of EEM-PARAFAC modeling provides insight into the composition and fate of agricultural DOM in natural and engineered systems.

Binding of Sulpiride to Seric Albumins

PubMed Central

da Silva Fragoso, Viviane Muniz; de Morais Coura, Carla Patrícia; Hoppe, Luanda Yanaan; Soares, Marília Amável Gomes; Silva, Dilson; Cortez, Celia Martins

2016-01-01

The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride–HSA were 2.20 (±0.08) × 104 M−1, at 37 °C, and 5.46 (±0.20) × 104 M−1, at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 104 M−1, at 37 °C and 2.17 (±0.04) × 104 M−1, at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure. PMID:26742031

Excitation-emission matrices and synchronous fluorescence spectroscopy for the diagnosis of gastrointestinal cancers

NASA Astrophysics Data System (ADS)

Genova, Ts; Borisova, E.; Penkov, N.; Vladimirov, B.; Zhelyazkova, A.; Avramov, L.

2016-06-01

We report the development of an improved fluorescence technique for cancer diagnostics in the gastrointestinal tract. We investigate the fluorescence of ex vivo colorectal (cancerous and healthy) tissue samples using excitation-emission matrix (EEM) and synchronous fluorescence spectroscopy (SFS) steady-state approaches. The obtained results are processed for revealing characteristic fluorescence spectral features with a valuable diagnostic meaning. The main tissue fluorophores, contributing to the observed fluorescence, are tyrosine, tryptophan, NADH, FAD, collagen and elastin. Based on the results of the Mann-Whitney test as useful parameters for differentiation of gastrointestinal cancer from normal mucosa, we suggest using excitation wavelengths in the range 300 - 360 nm for fluorescence spectroscopy and wavelengths intervals of 60 nm and 90 nm for SFS.

Characterization of chromophoric dissolved organic matter (CDOM) in rainwater using fluorescence spectrophotometry.

PubMed

Salve, P R; Lohkare, H; Gobre, T; Bodhe, G; Krupadam, R J; Ramteke, D S; Wate, S R

2012-02-01

The fluorescence excitation-emission matrix of Chromophoric dissolved organic matter (CDOM) samples from rainwater collected at Rameswaram, Tamilnadu, India are analysed. Total five peaks were observed for humic/marine and protein likes substances respectively. The peak A and C intensities varies form 1.98 ± 0.28 and 0.97 ± 0.11 QSU respectively represents humic like substances. The peak B and T intensities varies from 3.94 ± 0.75 and 7.42 ± 1.43 QSU showed association of protein like substances whereas peak M intensities varies from 1.92 ± 0.37 QSU indicates marine contribution. Among the fluorophores, the following sequence were observed as T > B > A > M > C which indicates dominance of Tryptophan like substances in rainwater. The average peak T/C ratios was observed as 7.88 ± 2.2 indicates microbial contamination by Tryptophan-like substances with the high biological activity and low volatility.

[Photodegradation of chromophoric dissolved organic matter from Jiulong River Estuary under natural solar radiation].

PubMed

Guo, Wei-dong; Cheng, Yuan-yue

2008-06-01

Low salinity water sample collected from Jiulong River Estuary filtered using 0.2 microm Millipore filter was exposed to natural solar radiation from 10:00 to 16:00 each day during one week period in early and late May, 2005. Photodegradation of fluorescence and absorption properties of CDOM (chromophoric dissolved organic matter) was observed. The results showed that humic-like fluorescence (lambda Ex/lambda Em = 350/450 nm), tryptophan-like fluorescence (lambda Ex/lambda Em = 225/350 nm) and absorption coefficient of CDOM can be significantly photodegraded during short-term solar exposure in early summer. These photodegradation processes followed the first-order dynamic equation. The degradation half time of humic-like fluorescence, tryptophan-like fluorescence and a (280) were calculated as 3.5-5.1 d, 3.0-4.5 d and 6.3 d. The absorption loss spectra of CDOM indicated that the solar UV radiation was responsible for the photochemical degradation of CDOM. The loss of humic-like fluorescence (70%) was obviously higher than loss of a (280) (about 40%), suggesting that photobleaching ability of CDOM fluorophores were much stronger than CDOM chromophores. However, the correlation relationship between humic-like fluorescence and absorption coefficient are still kept. A250/A350 of CDOM increased till the end of radiation experiment compared with the control group, suggesting photodegradation may decrease the average molecular size of CDOM. These findings show that terrestrial CDOM can be transformed and removed by photochemical decomposition after transport into the sea, and photodegradation might be an important sink for terrestrial CDOM.

Characterization of CDOM from urban waters in Northern-Northeastern China using excitation-emission matrix fluorescence and parallel factor analysis.

PubMed

Zhao, Ying; Song, Kaishan; Li, Sijia; Ma, Jianhang; Wen, Zhidan

2016-08-01

Chromophoric dissolved organic matter (CDOM) plays an important role in aquatic systems, but high concentrations of organic materials are considered pollutants. The fluorescent component characteristics of CDOM in urban waters sampled from Northern and Northeastern China were examined by excitation-emission matrix fluorescence and parallel factor analysis (EEM-PARAFAC) to investigate the source and compositional changes of CDOM on both space and pollution levels. One humic-like (C1), one tryptophan-like component (C2), and one tyrosine-like component (C3) were identified by PARAFAC. Mean fluorescence intensities of the three CDOM components varied spatially and by pollution level in cities of Northern and Northeastern China during July-August, 2013 and 2014. Principal components analysis (PCA) was conducted to identify the relative distribution of all water samples. Cluster analysis (CA) was also used to categorize the samples into groups of similar pollution levels within a study area. Strong positive linear relationships were revealed between the CDOM absorption coefficients a(254) (R (2) = 0.89, p < 0.01); a(355) (R (2) = 0.94, p < 0.01); and the fluorescence intensity (F max) for the humic-like C1 component. A positive linear relationship (R (2) = 0.77) was also exhibited between dissolved organic carbon (DOC) and the F max for the humic-like C1 component, but a relatively weak correlation (R (2) = 0.56) was detected between DOC and the F max for the tryptophan-like component (C2). A strong positive correlation was observed between the F max for the tryptophan-like component (C2) and total nitrogen (TN) (R (2) = 0.78), but moderate correlations were observed with ammonium-N (NH4-N) (R (2) = 0.68), and chemical oxygen demand (CODMn) (R (2) = 0.52). Therefore, the fluorescence intensities of CDOM components can be applied to monitor water quality in real time compared to that of traditional approaches. These results demonstrate that EEM-PARAFAC is useful to evaluate the dynamics of CDOM fluorescent components in urban waters from Northern and Northeastern China and this method has potential applications for monitoring urban water quality in different regions with various hydrological conditions and pollution levels.

Spectroscopic Study of the Interaction between Horse Heart Myoglobin and Zirconium(IV)-Substituted Polyoxometalates as Artificial Proteases.

PubMed

Ly, Hong Giang T; Parac-Vogt, Tatjana N

2017-09-20

A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and 1 H and 31 P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 Zr IV -Keggin, exhibits the strongest interaction with HHM. 31 P NMR spectroscopy studies show that this POM dissociates in solution, resulting in the formation of a monomeric 1:1 Zr IV -Keggin structure, which is likely to be a catalytically active species. In the presence of Zr IV -POMs, HHM does not undergo complete denaturation, as evidenced by CD, UV/Vis, tryptophan fluorescence, and 1 H NMR spectroscopy. CD spectroscopy shows a gradual decrease in the α-helical content of HHM upon addition of Zr IV -POMs. The largest effect is observed in the presence of a large Zr IV -Wells-Dawson structure, whereas small Zr IV -Lindqvist POM has the least influence on the decrease in the α-helical content of HHM. In all cases, the Soret band at λ=409 nm is maintained in the presence of all examined Zr-POMs, which indicates that no conformational changes in the protein occur near the heme group. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

PubMed Central

Fernandes, Carlos A. H.; Pazin, Wallance M.; Dreyer, Thiago R.; Bicev, Renata N.; Cavalcante, Walter L. G.; Fortes-Dias, Consuelo L.; Ito, Amando S.; Oliveira, Cristiano L. P.; Fernandez, Roberto Morato; Fontes, Marcos R. M.

2017-01-01

Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation. PMID:28256632

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism.

PubMed

Fernandes, Carlos A H; Pazin, Wallance M; Dreyer, Thiago R; Bicev, Renata N; Cavalcante, Walter L G; Fortes-Dias, Consuelo L; Ito, Amando S; Oliveira, Cristiano L P; Fernandez, Roberto Morato; Fontes, Marcos R M

2017-03-03

Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A 2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

[The Influence of Runoff Pollution to DOM Features in an Urban Wastewater Treatment Plant].

PubMed

He, Li; Ji, Fang-ying; Lai, Ming-sheng; Xu, Xuan; Zhou, Wei-wei; Mao, Bo-lin; Yang, Ming-jia

2015-03-01

Combined with wastewater treatment process, the sewage in sunny and rainy day was collected from a wastewater treatment plant in Chongqing. Three-dimensional fluorescence spectra was used to investigate the characteristic fluorescence of dissolved organic matter (DOM). DOM dissolved organic carbon (DOC), chemical oxygen demand (COD), fluorescence index (ƒ450/500) and fluorescence intensity ratio γ (A, C) of fulvic acid in ultraviolet and visible region were used to analyze the impact of rain runoff pollution on sewage DOM. According to the experimental data, the DOM fluorescence fingerprints of this wastewater treatment plant were quite different from typical municipal sewage, and the main component was tryptophan with low excitation wavelength (Peak S), then the tryptophan with long wavelength excitation (Peak T) followed. A2/O process had an approximative degradation of the protein-like both in sunny day and rainy day, but had a better degradation of fulvic-like, DOC and COD in rainy day than that in sunny day. Morever, the fluorescence peaks got red-shifted after the biological treatment. The differences of DOM fluorescence fingerprint between sunny and rainy day were significant, the fluorescence center of UV fulvic (Peak A) in rainy day getting blue-shifted obviously, shifting from 240 - 248/390 - 440 to 240 - 250/370 - 400 nm. Although the DOM types in sunny and rainy day were the same, the source of fulvic got more complex by runoff and the component ratio of DOM also changed. Compared with the sunny day, the proportion of Peak S in DOM dereased by 10%, and the proportion of Peak A increased by 7% in rainy day.

Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

NASA Astrophysics Data System (ADS)

Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

2011-04-01

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-L-Levodopa Schiff Bases with bovine serum albumin (BSA).

PubMed

Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

2011-04-01

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-D-Phenylalanine (o-VDP), o-Vanillin-L-Tyrosine (o-VLT) and o-Vanillin-L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant (K(q)), apparent quenching constant (K(sv)), effective binding constant (K(A)) and corresponding dissociation constant (K(D)) as well as binding site number (n) were obtained. In addition, the binding distance (r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA>o-VLT-BSA>o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues. Copyright © 2010 Elsevier B.V. All rights reserved.

Interactions of the EcoRV restriction endonuclease with fluorescent oligodeoxynucleotides.

PubMed

Erskine, S G; Halford, S E

1995-05-19

A self-complementary dodecadeoxyribonucleotide that contains the recognition sequence for the R.EcoRV ENase was synthesised with a primary amino group at its 5' terminus. The 5' amino function was labeled with the fluorescent dye 5-[dimethylamino] napthalene-1-sulfonyl chloride. The labeled oligodeoxyribonucleotide in its duplex form was shown to be a suitable substrate for kinetic studies on the ENase and that no significant dye-DNA or dye-protein interactions occurred. Finally, the binding of R.EcoRV to the labeled DNA was followed by detecting the fluorescence resonance energy transfer between the tryptophans of the protein and the fluorescent labels of the DNA.

Stability improvement of natural food colors: Impact of amino acid and peptide addition on anthocyanin stability in model beverages.

PubMed

Chung, Cheryl; Rojanasasithara, Thananunt; Mutilangi, William; McClements, David Julian

2017-03-01

Anthocyanins are prone to chemical degradation and color fading in the presence of vitamin C. The potential of three amino acids (l-phenylalanine, l-tyrosine, l-tryptophan) and a polypeptide (ε-poly-l-lysine) in prolonging the color stability of purple carrot anthocyanins (0.025%) in model beverages (0.05% l-ascorbic acid, citric acid, pH 3.0) stored at elevated temperature (40°C/7 days) was examined. In the absence of amino acids or peptides, anthocyanin degraded at first-order reaction rate. Addition of amino acids or peptide (0.1%) increased the color stability of anthocyanins, with the most significant improvement observed for l-tryptophan. The average half-life of anthocyanin color increased from 2 days to 6 days with l-tryptophan addition. Fluorescence quenching measurements revealed that the l-tryptophan interacted with anthocyanins mainly through hydrogen bonding, although some hydrophobic interaction may also have been involved. Overall, this study suggests that amino acid or peptide addition may prolong the color stability of anthocyanin in beverage products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Copper nanocluster-enhanced luminol chemiluminescence for high-selectivity sensing of tryptophan and phenylalanine.

PubMed

Borghei, Yasaman-Sadat; Hosseini, Morteza; Khoobi, Mehdi; Ganjali, Mohammad Reza

2017-09-01

A remarkable method for the highly sensitive detection of phenylalanine and tryptophan based on a chemiluminescence (CL) assay was reported. It was found that fluorescent copper nanoclusters capped with cysteine (Cys-CuNCs) strongly enhance the weak CL signal resulting from the reaction between luminol and H 2 O 2 . Of the amino acids tested, phenylalanine and tryptophan could enhance the above CL system sensitively. Under optimum conditions, this method was satisfactorily described by a linear calibration curve over a range of 1.0 × 10 -6 to 2.7 × 10 -5  M for phenylalanine and 1.0 × 10 -7 to 3.0 × 10 -5  M for tryptophan, respectively. The effect of various parameters such as Cys-CuNC concentration, H 2 O 2 concentration and pH on the intensity of the CL system were also studied. The main experimental advantage of the proposed method was its selectivity for two amino acids compared with others. To evaluate the applicability of the method to the analysis of a real biological sample it was used to determine tryptophan and phenylalanine in human serum and remarkable results were obtained. Copyright © 2017 John Wiley & Sons, Ltd.

The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

PubMed Central

McIntyre, J C; Hundley, P; Behnke, W D

1987-01-01

Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52. PMID:3663193

Spectroscopic investigation of the interaction of water-soluble azocalix[4]arenes with bovine serum albumin.

PubMed

Fan, Ping; Wan, Lu; Shang, Yunshan; Wang, Jun; Liu, Yulong; Sun, Xiaoyu; Chen, Chen

2015-02-01

In this work, three hydrosoluble azocalix[4]arene derivatives, 5-(o-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (o-MAC-Calix), 5-(m-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (m-MAC-Calix) and 5-(p-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (p-MAC-Calix) were synthesized. Their structures were characterized by infrared spectrum (IR), nuclear magnetic resonance spectrum (1H NMR and 13C NMR) and mass spectrum (MS). The interactions between these compounds and bovine serum albumin (BSA) were studied by fluorescence spectroscopy, UV-vis spectrophotometry and circular dichroic spectroscopy. According to experimental results, three azocalix[4]arene derivatives can efficiently bind to BSA molecules and the o-MAC-Calix displays more efficient interactions with BSA molecules than m-MAC-Calix and p-MAC-Calix. Molecular docking showed that the o-MAC-Calix was embedded in the hydrophobic cavity of helical structure of BSA molecular and the tryptophan (Trp) residue of BSA molecular had strong interaction with o-MAC-Calix. The fluorescence quenching of BSA caused by azocalix[4]arene derivatives is attributed to the static quenching process. In addition, the synchronous fluorescence spectroscopy indicates that these azocalix[4]arene derivatives are more accessible to Trp residues of BSA molecules than the tyrosine (Tyr) residues. The circular dichroic spectroscopy further verified the binding of azocalix[4]arene derivatives and BSA. Copyright © 2014 Elsevier Inc. All rights reserved.

The role of protein characteristics in the formation and fluorescence of Au nanoclusters

NASA Astrophysics Data System (ADS)

Xu, Yaolin; Sherwood, Jennifer; Qin, Ying; Crowley, Dorothy; Bonizzoni, Marco; Bao, Yuping

2014-01-01

Protein-encapsulated gold nanoclusters have shown many advantages over other gold nanocluster systems, including green synthesis, biocompatibility, high water solubility, and the ease of further conjugation. In this article, we systematically investigated the effects of the protein size and amino acid content on the formation and fluorescent properties of gold nanoclusters using four model proteins (bovine serum albumin, lysozyme, trypsin, and pepsin). We discovered that the balance of amine and tyrosine/tryptophan containing residues was critical for the nanocluster formation. Protein templates with low cysteine contents caused blue shifts in the fluorescent emissions and difference in fluorescent lifetimes of the gold nanoclusters. Furthermore, the protein size was found to be a critical factor for the photostability and long-term stability of gold nanoclusters. The size of the protein also affected the Au nanocluster behaviour after immobilization.Protein-encapsulated gold nanoclusters have shown many advantages over other gold nanocluster systems, including green synthesis, biocompatibility, high water solubility, and the ease of further conjugation. In this article, we systematically investigated the effects of the protein size and amino acid content on the formation and fluorescent properties of gold nanoclusters using four model proteins (bovine serum albumin, lysozyme, trypsin, and pepsin). We discovered that the balance of amine and tyrosine/tryptophan containing residues was critical for the nanocluster formation. Protein templates with low cysteine contents caused blue shifts in the fluorescent emissions and difference in fluorescent lifetimes of the gold nanoclusters. Furthermore, the protein size was found to be a critical factor for the photostability and long-term stability of gold nanoclusters. The size of the protein also affected the Au nanocluster behaviour after immobilization. Electronic supplementary information (ESI) available See DOI: 10.1039/c3nr06040c

Glycation of human lens proteins: preferential glycation of alpha A subunits.

PubMed

Swamy, M S; Abraham, A; Abraham, E C

1992-03-01

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16-85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg-1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg-1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of alpha-crystallin, while the increases in beta and gamma were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10-50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

PubMed

Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

2000-12-01

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

PubMed Central

Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

2000-01-01

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed. PMID:11085949

Fluorescence studies on the interaction of choline-binding domain B of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Damai, Rajani S; Anbazhagan, V; Rao, K Babu; Swamy, Musti J

2009-12-01

The microenvironment and accessibility of the tryptophan residues in domain B of PDC-109 (PDC-109/B) in the native state and upon ligand binding have been investigated by fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) studies. The increase in the intrinsic fluorescence emission intensity of PDC-109/B upon binding to lysophosphatidylcholine (Lyso-PC) micelles and dimyristoylphosphatidylcholine (DMPC) membranes was considerably less as compared to that observed with the whole PDC-109 protein. The degree of quenching achieved by different quenchers with PDC-109/B bound to Lyso-PC and DMPC membranes was significantly higher as compared to the full PDC-109 protein, indicating that membrane binding afforded considerably lesser protection to the tryptophan residues of domain B as compared to those in the full PDC-109 protein. Finally, changes in red-edge excitation shift (REES) seen with PDC-109/B upon binding to DMPC membranes and Lyso-PC micelles were smaller that the corresponding changes in the REES values observed for the full PDC-109. These results, taken together suggest that intact PDC-109 penetrates deeper into the hydrophobic parts of the membrane as compared to domain B alone, which could be the reason for the inability of PDC-109/B to induce cholesterol efflux, despite its ability to recognize choline phospholipids at the membrane surface.

Trials and Tribulations of Fluorescent Dissolved Organic Matter Chemical Interpretations: A case study of polar ice cores

NASA Astrophysics Data System (ADS)

D'Andrilli, J.

2017-12-01

Excitation emission matrix fluorescence spectroscopy is widely applied for rapid dissolved organic matter (DOM) characterization in aquatic systems. Fluorescent DOM surveys are booming, not only as a central focus in aquatic environments, but also as an important addition to interdisciplinary research (e.g., DOM analysis in concert with ice core paleoclimate reconstructions, stream metabolism, hydrologic regimes, agricultural developments, and biological activity), opening new doors, not just for novelty, but also for more challenges with chemical interpretations. Recently, the commonly used protein- versus humic-like classifications of DOM have been ineffective at describing DOM chemistry in various systems (e.g., ice cores, wastewaters, incubations/engineered). Moreover, the oversimplification of such classifications used to describe fluorescing components, without further scrutiny, has become commonplace, ultimately producing vague reporting. For example, West Antarctic ice core DOM was shown to contain fluorescence in the low excitation/emission wavelength region, however resolved fluorophores depicting tyrosine- and tryptophan-like DOM were not observed. At first, as literature suggested, we reported this result as protein-like, and concluded that microbial contributions were dominant in deep ice. That initial interpretation would disintegrate the conservation paradigm of atmospheric composition during deposition, the crux of ice core research, and contradict other lines of evidence. This begged the question, "How can we describe DOM chemistry without distinct fluorophores?" Antarctic ice core DOM was dominated by neither tyrosine- nor tryptophan-like fluorescence, causing "unusual" looking fluorescent components. After further examination, deep ice DOM was reported to contain fluorescent species most similar to monolignols and tannin-like phenols, describing the precursors of lignin from low carbon producing environments, consistent with marine sediment records. Currently, we are working towards more detailed descriptions of fluorescence, thus accepting variation in and around protein- and humic-like regions, and achieving robust chemical interpretations of DOM chemistry, ultimately providing insight to its interwoven nature in the environment.

pH-insensitive electrostatic interaction of carmoisine with two serum proteins: a possible caution on its uses in food and pharmaceutical industry.

PubMed

Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

2013-07-05

Here we have investigated the binding of carmoisine, a water-soluble azo food colorant, with serum proteins (HSA and BSA) by fluorescence and UV-VIS spectroscopy, circular dichroism and molecular docking studies. Results indicate that fluorescence quenching of protein has been due to site-specific binding of the dye with biomacromolecules. Site marker competitive binding and molecular docking explorations show that interaction occurs in the sub-domain ІІA of HSA and the sub-domains ІІA and ІB in the case of BSA. Conformational investigation indicates that dye binding modifies the secondary structure of proteins and this also alters the microenvironment of the tryptophan(s). The interaction is found to be pH-insensitive which can have relevance to the toxicological profiles of the dye, and ionic strength dependence of binding can be exploited in protein purification mediated by such food colorants. Copyright © 2013 Elsevier B.V. All rights reserved.

Quenching interaction of BSA with DTAB is dynamic in nature: A spectroscopic insight

NASA Astrophysics Data System (ADS)

Das, Nirmal Kumar; Pawar, Lavanya; Kumar, Naveen; Mukherjee, Saptarshi

2015-08-01

The role of electrostatic interactions between the protein, Bovine Serum Albumin (BSA) and the cationic surfactant, dodecyltrimethylammonium bromide (DTAB) has been substantiated using spectroscopic approaches. The primary mechanism of fluorescence quenching of the tryptophan of BSA is most probably dynamic in nature as the complex formation resulting in a protein-surfactant assembly is not very spontaneous. The weak interaction buries the tryptophan amino acid residue inside the protein scaffolds which have been quantitatively proved by our acrylamide quenching studies. The loss in the secondary structure of the protein as a result of interaction with DTAB has been elucidated by CD spectroscopy.

Luminescence diagnostics of conformational changes of the Hsp70 protein in the course of thermal denaturation

NASA Astrophysics Data System (ADS)

Bukina, M. N.; Bakulev, V. M.; Barmasov, A. V.; Zhakhov, A. V.; Ishchenko, A. M.

2015-06-01

The spectral luminescence properties of aqueous solutions of the Hsp70 protein are studied, the dependence of the luminescence spectrum on the excitation wavelength is revealed, and the temperature dependence of luminescence intensity of tyrosine and tryptophan residues in the temperature interval of 20-80° C is analyzed. The luminescence method is used to determine temperature interval (42-57° C) in which protein melting takes place. An increase in the fluorescence quantum yield of tryptophan and the bathochromic shift of the emission spectrum of denatured Hsp70 prove that transition takes place of tryptophanyls to the surface of the protein molecule.

Metallic-nanoparticles-enhanced fluorescence from individual micron-sized aerosol particles on-the-fly.

PubMed

Sivaprakasam, Vasanthi; Hart, Matthew B; Jain, Vaibhav; Eversole, Jay D

2014-08-11

Fluorescence spectra from individual aerosol particles that were either coated or embedded with metallic nanoparticles (MNPs) was acquired on-the-fly using 266 nm and 355 nm excitation. Using aqueous suspensions of MNPs with either polystyrene latex (PSL) spheres or dissolved proteins (tryptophan or ovalbumin), we generated PSL spheres coated with MNPs, or protein clusters embedded with MNPs as aerosols. Both enhanced and quenched fluorescence intensities were observed as a function of MNP concentration. Optimizing MNP material, size and spacing should yield enhanced sensitivity for specific aerosol materials that could be exploited to improve detection limits of single-particle, on-the-fly fluorescence or Raman based spectroscopic sensors.

Distinct Kynureninase and Hydroxykynureninase Activities in Microorganisms: Occurrence and Properties of a Single Physiologically Discrete Enzyme in Yeast

PubMed Central

Shetty, A. S.; Gaertner, F. H.

1973-01-01

(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l-tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l-tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l-tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa, an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l-3-hydroxykynurenine and l-kynurenine of 6.7 × 10−6 and 5.4 × 10−4 M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa. (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals. PMID:4266242

X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging.

PubMed

Malo, Gabrielle D; Pouwels, Lauren J; Wang, Meitian; Weichsel, Andrzej; Montfort, William R; Rizzo, Mark A; Piston, David W; Wachter, Rebekka M

2007-09-04

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.

A new fluorescent and electrochemical Zn2+ ion sensor based on Schiff base derived from benzil and L-tryptophan.

PubMed

Dutta, Kaku; Deka, Ramesh C; Das, Diganta Kumar

2014-04-24

Single molecule acting as both fluorescent and electrochemical sensor for Zn(2+) ion is rare. The product (L) obtained on condensation between benzil and L-tryptophan has been characterized by H NMR, ESI-MS and FT-IR spectroscopy. L in 1:1 (v/v) CH3OH:H2O solution shows fluorescence emission in the range 300 nm to 600 nm with λmax at 350 nm when is excited with 295 nm photon. Zn(2+) ion could induce a 10-fold enhancement in fluorescent intensity of L. Fluorescence and UV/Visible spectral data analysis shows that the binding ratio between Zn(2+) ion and L is 1:1 with log β=4.55. Binding of Zn(2+) ion disrupts the photoinduced electron transfer (PET) process in L and causes the fluorescence intensity enhancement. When cyclic voltammogram is recorded for L in 1:1 (v/v) CH3OH:H2O using glassy carbon (GC) electrode, two quasi reversible redox couples at redox potential values -0.630±0.005 V and -1.007±0.005 V are obtained (Ag-AgCl as reference, scan rate 0.1 V s(-1)). Interaction with Zn(2+) ion makes the first redox couple irreversible while the second couple undergoes a 0.089 V positive shift in redox potential. Metal ions - Cd(2+), Cu(2+), Co(2+), Hg(2+), Ag(+), Ni(2+), Fe(2+), Mn(2+), Mg(2+), Ca(2+)and Pb(2+), individually or all together, has no effect on the fluorescent as well as electrochemical property of L. DFT calculations showed that Zn(2+) ion binds to L to form a stable complex. The detection limit for both fluorescence as well as electrochemical detection was 10(-6) M. Copyright © 2013 Elsevier B.V. All rights reserved.

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.

PubMed

Baumann, Tobias; Schmitt, Franz-Josef; Pelzer, Almut; Spiering, Vivian Jeanette; Freiherr von Sass, Georg Johannes; Friedrich, Thomas; Budisa, Nediljko

2018-04-27

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Impacts of aeration management and polylactic acid addition on dissolved organic matter characteristics in intensified aquaponic systems.

PubMed

Wu, Haiming; Zou, Yina; Lv, Jialong; Hu, Zhen

2018-08-01

Aquaponics as a potential alternative for conventional aquaculture industry has increasingly attracted worldwide attention in recent years. However, the sustainable application of aquaponics is facing a growing challenge. In particular, there is a pressing need to better understand and control the accumulation of dissolved organic matter (DOM) in aquaponics with the aim of optimizing nitrogen utilization efficiency. This study was aiming for assessing the characteristics of DOM in the culture water and the relationship with the nitrogen transformations in different intensified aquaponic systems with hydroponic aeration supplement and polylactic acid (PLA) addition. Two enhancing attempts altered the quantity of DOM in aquaponic systems significantly with a varying DOM content of 21.98-45.65 mg/L. The DOM could be represented by four identified fluorescence components including three humic -like materials (83-86%) and one tryptophan-like substance (14-17%). The fluorescence intensities of humic acid-like components were decreased significantly after the application of intensifying strategies, which indicating that two enhancing attempts possibly affected humic acid-like fluorescence. Variation of optical indices also suggested the reductions of water DOM which could be impacted by the enhancing nitrogen treatment processes. These findings will benefit the potential applications and sustainable operation of these strategies in aquaponics. Copyright © 2018 Elsevier Ltd. All rights reserved.

Deciphering the toxicity of bisphenol a to Candida rugosa lipase through spectrophotometric methods.

PubMed

Zhang, Rui; Zhao, Lining; Liu, Rutao

2016-10-01

Bisphenol A is widely used in the manufacture of food packaging and beverage containers and can invade our food and cause contamination. Candida rugose lipase has been a versatile enzyme for biocatalysis and biotransformations to produce useful materials for food, pharmaceutical and flavor. The interactions between bisphenol A and Candida rugosa lipase in vitro were studied by UV-vis, steady-state fluorescence, circular dichroism, synchronous fluorescence, light scattering spectra, molecular docking and enzyme activity assay to better understand the toxicity and toxic mechanisms of bisphenol A. The intrinsic fluorescence of the tryptophan amino acid residue and the secondary structure of the globular protein candida rugose lipase were made use of to thoroughly investigate the structural changes caused by bisphenol A. The results of the fluorescence indicated that bisphenol A interacted with candida rugose lipase and made tryptophan be exposed to a hydrophobic environment. Multi-spectroscopic measurements showed that the addition of bisphenol A increased the intrinsic fluorescence of Candida rugosa lipase, loosened its skeleton structure and changed its secondary structure. Also, the increased activity of Candida rugosa lipase revealed that the position or the structure of the catalytic triad of Candida rugosa lipase may be changed. The molecular docking results showed that bisphenol A bound with the residue Serine 209 which could be another reason for the increased activity of Candida rugosa lipase. Moreover, as can be seen from the results of resonance light scattering and dynamic light scattering, the volume of the Candida rugosa lipase was decreased and the lid may be stripped. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessment of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge: Extracellular polymeric substances contribution and soluble microbial products release.

PubMed

Huang, Linxian; Li, Meilin; Si, Guangchao; Wei, Jinglin; Ngo, Huu Hao; Guo, Wenshan; Xu, Weiying; Du, Bin; Wei, Qin; Wei, Dong

2018-05-18

In the present study, the responses of microbial products in the biosorption process of Cu(II) onto aerobic granular sludge were evaluated by using batch and spectroscopic approaches. Batch experimental data showed that extracellular polymeric substances (EPSs) contributed to Cu(II) removal from an aqueous solution, especially when treating low metal concentrations, whereas soluble microbial products (SMPs) were released under the metal stress during biosorption process. A three-dimensional excitation-emission matrix (3D-EEM) identified four main fluorescence peaks in the EPS, i.e., tryptophan protein-like, aromatic protein-like, humic-like and fulvic acid-like substances, and their fluorescence intensities decreased gradually in the presence of Cu(II) during the sorption process. Particularly, tryptophan protein-like substances quenched the Cu(II) binding to a much higher extent through a static quenching process with less than one class of binding sites. According to the synchronous fluorescence spectra, the whole fluorescence intensity of released SMP samples expressed an increased trend with different degrees along with contact time. Two-dimensional correlation spectroscopy (2D-COS) suggested that the fulvic-like fluorescence fraction might be more susceptible to metal exposure than other fractions. The result of molecular weight distribution demonstrated that the SMPs released from the biosorption process differed significantly according to contact time. The result obtained could provide new insights into the responses of microbial products from aerobic granular sludge with heavy metal treatment. Copyright © 2018. Published by Elsevier Inc.

Tryptophan-to-Tryptophan Energy Transfer in UV-B photoreceptor UVR8

NASA Astrophysics Data System (ADS)

Li, Xiankun; Zhong, Dongping

UVR8 (UV RESISTANCE LOCUS 8) protein is a UV-B photoreceptor in high plants. UVR8 is a homodimer that dissociates into monomers upon UV-B irradiation (280 nm to 315 nm), which triggers various protective mechanisms against UV damages. Uniquely, UVR8 does not contain any external chromophores and utilizes the UV-absorbing natural amino acid tryptophan (Trp) to perceive UV-B. Each UVR8 monomer has 14 tryptophan residues. However, only 2 epicenter Trp (W285 W233) are critical to the light induced dimer-to-monomer transformation. Here, we revealed, using site-directed mutagenesis and spectroscopy, a striking energy flow network, in which other tryptophan chromophores serve as antenna to transfer excitation energy to epicenter Trp, greatly enhancing UVR8 light-harvesting efficiency. Furthermore, Trp-to-Trp energy transfer rates were measured and agree well with theoretical values.

Effect of perfluorooctane sulfonate on the conformation of wheat germ acid phosphatase.

PubMed

Xu, Dongmei; Jin, Jianchang; Shen, Tong; Wang, Yanhua

2013-11-01

Fluorescence spectroscopy was used to study the quenching mechanism, the type of force and the binding sites of perfluorooctane sulfonate (PFOS) on wheat germ acid phosphatase (ACPase). The results showed that the quenching effect of PFOS on ACPase was mainly due to a static quenching mechanism that occurred via the formation of hydrogen bonds and van der Waals forces. The results from synchronous fluorescence spectroscopy demonstrated that PFOS interacts with ACPase close to the tryptophan residues. In addition, synchronous fluorescence spectroscopy also showed that PFOS increases the hydrophobicity of the microenvironment of the tyrosine residues, hence decreasing the local polarity.

Fluorescence of bioaerosols: mathematical model including primary fluorescing and absorbing molecules in bacteria.

PubMed

Hill, Steven C; Pan, Yong-Le; Williamson, Chatt; Santarpia, Joshua L; Hill, Hanna H

2013-09-23

This paper describes a mathematical model of fluorescent biological particles composed of bacteria, viruses, or proteins. The fluorescent and/or light absorbing molecules included in the model are amino acids (tryptophan, etc.); nucleic acids (DNA, RNA, etc.); coenzymes (nicotinamide adenine dinucleotides, flavins, and vitamins B₆ and K and variants of these); and dipicolinates. The concentrations, absorptivities, and fluorescence quantum yields are estimated from the literature, often with large uncertainties. The bioparticles in the model are spherical and homogeneous. Calculated fluorescence cross sections for particles excited at 266, 280, and 355 nm are compared with measured values from the literature for several bacteria, bacterial spores and albumins. The calculated 266- and 280-nm excited fluorescence is within a factor of 3.2 of the measurements for the vegetative cells and proteins, but overestimates the fluorescence of spores by a factor of 10 or more. This is the first reported modeling of the fluorescence of bioaerosols in which the primary fluorophores and absorbing molecules are included.

Interactions between α-amylase and an acidic branched polysaccharide from green tea.

PubMed

Wu, Shuyun; Lai, Minghua; Luo, Jiahao; Pan, Jingwen; Zhang, Li-Ming; Yang, Liqun

2017-01-01

To understand the mechanism responsible for the α-amylase inhibitory activity of tea polysaccharides, the interaction between α-amylase and an acidic branched tea polysaccharide (TPSA) was investigated using fluorescence spectroscopy and resonance light scattering analysis. TPSA, exhibiting inhibitory activity towards α-amylase (the maximum inhibition percentage of 65%), was isolated from green tea (Camellia sinensis) and characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and gas chromatography. Synchronous fluorescence spectroscopy revealed that the binding interaction between the tryptophan residues of α-amylase and TPSA was predominant. Based on the fluorescence quenching effect of tryptophan residues induced by TPSA, the binding constants between α-amylase and TPSA were determined to be 18.6×10 6 , 8.0×10 6 and 4.6×10 6 L·mol -1 at 20, 30 and 37°C, respectively. The calculated Gibbs free-energy changes were negative, indicating that the bonding interaction was a spontaneous process. The enthalpy and the entropy changes were -62.13 KJ·mol -1 and -0.0728 KJ·mol -1 ·K -1 , suggesting that hydrogen bonding interactions might play a major role in the binding process. The formation of an α-amylase/TPSA complex was evidenced by fluorescence quenching and resonance light scattering analysis, and this complex could be the main contributor to the α-amylase inhibitory activity of TPSA. Copyright © 2016 Elsevier B.V. All rights reserved.

The Fluorescent Properties of Dissolved Organic Matter and Assessment of Total Nitrogen in Overlying Water with Different Dissolved Oxygen Conditions.

PubMed

Zhang Hua; Kuan, Wang; Song, Jian; Zhang, Yong; Huang, Ming; Huang, Jian; Zhu, Jing; Huang, Shan; Wang, Meng

2016-03-01

This paper used excitation-emission matrix spectroscopy (EEMs) to probe the fluorescence properties of dissolved organic matter (DOM) in the overlying water with different dissolved oxygen (DO) conditions, investigating the relationship between protein-like fluorescence intensity and total nitrogen concentration. The resulting fluorescence spectra revealed three protein-like components (high-excitation wavelength tyrosine, low-excitation wavelength tyrosine, low-excitation wavelength tryptophan) and two fulvic-like components (ultraviolet fulvic-like components, visible fulvic-like components) in the overlying water. Moreover, the protein-like components were dominant in the overlying water's DOM. The fluorescence intensity of the protein-like components decreased significantly after aeration. Two of the protein-like components--the low-excitation wavelength tyrosine and the low-excitation wavelength tryptophan--were more susceptible to degradation by microorganisms within the degradable organic matter with respect to the high-excitation wavelength tyrosine. In contrast, the ultraviolet and visible fulvic-like fluorescence intensity increased along with increasing DO concentration, indicating that the fulvic-like components were part of the refractory organics. The fluorescence indices of the DOM in the overlying water were between 1.65-1.80, suggesting that the sources of the DOM were related to terrigenous sediments and microbial metabolic processes, with the primary source being the contribution from microbial metabolism. The fluorescence indices increased along with DO growth, which showed that microbial biomass and microbial activity gradually increased with increasing DO while microbial metabolism also improved, which also increased the biogenic components in the overlying water. The fluorescence intensity of the high-excitation wavelength tyrosine peak A showed a good linear relationship with the total nitrogen concentration at higher DO concentrations of 2.5, 3.5, and 5.5 mg x L(-1), with r2 being 0.956, 0.946, and 0.953, respectively. This study demonstrated that excitation-emission matrix spectroscopy can distinguish the transformation characteristics of the DOM and identify the linear relationship between the fluorescence intensity of the high-excitation wavelength tyrosine peak A and total nitrogen concentration, thus providing a quick and effective technique and theoretical support for river water monitoring and water restoration.

Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

PubMed Central

Robinson, Kirsten E.; Orans, Jillian; Kovach, Alexander R.; Link, Todd M.; Brennan, Richard G.

2014-01-01

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus. PMID:24288369

The influence of visible light and inorganic pigments on fluorescence excitation emission spectra of egg-, casein- and collagen-based painting media

NASA Astrophysics Data System (ADS)

Nevin, A.; Anglos, D.; Cather, S.; Burnstock, A.

2008-07-01

Spectrofluorimetric analysis of proteinaceous binding media is particularly promising because proteins employed in paintings are often fluorescent and media from different sources have significantly different fluorescence spectral profiles. Protein-based binding media derived from eggs, milk and animal tissue have been used for painting and for conservation, but their analysis using non-destructive techniques is complicated by interferences with pigments, their degradation and their low concentration. Changes in the fluorescence excitation emission spectra of films of binding media following artificial ageing to an equivalent of 50 and 100 years of museum lighting include the reduction of bands ascribed to tyrosine, tryptophan and Maillard reaction products and an increase in fluorescent photodegradation. Fluorescence of naturally aged paint is dependent on the nature of the pigment present and, with egg-based media, in comparison with un-pigmented films, emissions ascribed to amino acids are more pronounced.

Study on discrimination of oral cancer from normal using blood plasma based on fluorescence steady and excited state at excitation wavelength 280 nm

NASA Astrophysics Data System (ADS)

Rekha, Pachaiappan; Aruna, Prakasa Rao; Ganesan, Singaravelu

2016-03-01

Many research works based on fluorescence spectroscopy have proven its potential in the diagnosis of various diseases using the spectral signatures of the native key fluorophores such as tryptophan, tyrosine, collagen, NADH, FAD and porphyrin. These fluorophores distribution, concentration and their conformation may be changed depending upon the pathological and metabolic conditions of cells and tissues. In this study, we have made an attempt to characterize the blood plasma of normal subject and oral cancer patients by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed by employing the multivariate statistical method - linear discriminant analyses (LDA) using leaves one out cross validation method. The results illustrate the potential of fluorescence spectroscopy technique in the diagnosis of oral cancer using blood plasma.

Method of determining the optimal dilution ratio for fluorescence fingerprint of food constituents.

PubMed

Trivittayasil, Vipavee; Tsuta, Mizuki; Kokawa, Mito; Yoshimura, Masatoshi; Sugiyama, Junichi; Fujita, Kaori; Shibata, Mario

2015-01-01

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.

Fluorescent carbon dots as an efficient siRNA nanocarrier for its interference therapy in gastric cancer cells.

PubMed

Wang, Qing; Zhang, Chunlei; Shen, Guangxia; Liu, Huiyang; Fu, Hualin; Cui, Daxiang

2014-12-30

Fluorescent carbon dots (Cdots) have attracted increasing attention due to their potential applications in sensing, catalysis, and biomedicine. Currently, intensive research has been concentrated on the synthesis and imaging-guided therapy of these benign photoluminescent materials. Meanwhile, Cdots have been explored as nonviral vector for nucleic acid or drug delivery by chemical modification on purpose. We have developed a microwave assisted one-step synthesis of Cdots with citric acid as carbon source and tryptophan (Trp) as both nitrogen source and passivation agent. The Cdots with uniform size show superior water solubility, excellent biocompatibility, and high quantum yield. Afterwards, the PEI (polyethylenimine)-adsorbed Cdots nanoparticles (Cdots@PEI) were applied to deliver Survivin siRNA into human gastric cancer cell line MGC-803. The results have confirmed the nanocarrier exhibited excellent biocompatibility and a significant increase in cellular delivery of siRNA, inducing efficient knockdown for Survivin protein to 6.1%. In addition, PEI@Cdots complexes mediated Survivin silencing, the arrested cell cycle progression in G1 phase as well as cell apoptosis was observed. The Cdots-based and PEI-adsorbed complexes both as imaging agents and siRNA nanocarriers have been developed for Survivin siRNA delivery. And the results indicate that Cdots-based nanocarriers could be utilized in a broad range of siRNA delivery systems for cancer therapy.

Quenching of photoexcited states of the proteins chromophores and introduced into the protein macromolecules fluorescent probes by heavy metal ions

NASA Astrophysics Data System (ADS)

Melnikov, A. G.; Dyachuk, O. A.; Melnikov, G. V.

2015-03-01

We have studied the processes of quenching of photoexcited states of fluorescent probes and quenching of the fluorescence of the chromophores of human serum albumin (HSA) by heavy metal ions (HM): cations Tl+, Pb2+, Cu2+, Cd2+, and the anion of iodine (I-). We used the dye from xanthene series - eosin as a fluorescent probe. By quenching of the fluorescence of protein chromophores we found an influence of HM on the structure of proteins, resulting in a shift of the peak of the fluorescence of HSA tryptophanyl. This can be explained by proteins denaturation under the influence of heavy metals and penetration of water into the inner environment of HSA tryptophan. It was established that the constant of the quenching of the probe phosphorescence is much higher than the fluorescence, which is explained by significantly longer lifetime of the photoexcited states of fluorescent probes in the triplet state than in the singlet.

Fluorescence of Bacteria, Pollens, and Naturally Occurring Airborne Particles: Excitation/Emission Spectra

DTIC Science & Technology

2009-02-01

EEM spectra of Micrococcus lysodeikticus vegetative cells, dry............................... 24 Figure 18. EEM spectra of Micrococcus lysodeikticus...tryptophan, B. subtilis (vegetative cells) ATCC 6633, Pseudomonas fluorescens, Micrococcus lysodeikticus, Staphylococcus aureus, and Clostridium...present the EEM spectra of Pseudomonas fluorescens (dry), Yersinia pestis (dry and in water), Micrococcus lysodeikticus (dry and in water

pH-dependent differential interacting mechanisms of sodium dodecyl sulfate with bovine serum fetuin: a biophysical insight.

PubMed

Zaidi, Nida; Nusrat, Saima; Zaidi, Fatima Kamal; Khan, Rizwan H

2014-11-20

Sodium dodecyl sulfate (SDS)-glycoprotein interaction serves as a model for a biological membrane. To get mechanistic insight into the interaction of SDS and glycoprotein, the effect of SDS on bovine serum fetuin (BSF) was studied in subcritical micellar concentrations at pH 7.4 and pH 2 using multiple approaches. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically via its alkyl chains at pH 7.4 up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2, as confirmed by enhanced ThT fluorescence, β-sheet formation, and TEM microscopy, whereas BSF undergoes induction of an α-helical structure in the presence of higher SDS concentration at pH 7.4. The increase in α-helical content with increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of tryptophan conformers decreases, resulting in a decrement of the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

Plasmon resonance enhancement of nonlinear properties of amino acids

NASA Astrophysics Data System (ADS)

de Araujo, Renato E.; Rativa, Diego; Gomes, Anderson S. L.

2007-02-01

Here we analyze the influence of 9 nm (mean diameter) silver particles on the nonlinear properties of intrinsic cell molecules. A novel high sensitivity thermal managed eclipse Z-scan technique with a femtosecond laser system was used to analyze the nonlinear susceptibility of water solution of fluorescent and non-fluorescent amino acids (Tryptophan, Tyrosine, Phenylalanine, Proline and Histidine) with different concentration of silver nanoparticles. The generalized Maxwell Garnett model is used to explain the behavior of the measured nonlinear refractive index with the change of the nanoparticles concentration in the sample.

Application of portable in situ UV fluorescence sensors in natural and engineered aquatic systems.

NASA Astrophysics Data System (ADS)

Fox, Bethany; Rushworth, Cathy; Atrridge, John

2016-04-01

Natural organic matter (NOM) is ubiquitous throughout aquatic systems. This heterogeneous mixture of organic matter is central for aquatic ecosystems and, both local and global, biogeochemical cycling. Improvements in technology and data analysis has allowed for advances in the understanding and characterisation of aquatic organic matter. However, much of the technological expansions have focussed on benchtop instruments. In recent years, there has been interest in the continued development of portable in situ sensors for monitoring NOM characteristics within a wide range of applications, spanning both natural and engineered systems. The UviLux (Chelsea Technologies Group Ltd., UK) is an in situ portable UV fluorescence sensor that can be configured to monitor a range of NOM in aquatic systems, as well as anthropogenic inputs such as polycyclic aromatic hydrocarbons (PAH) and optical brighteners. Here we will focus on the use of the Tryptophan and CDOM UviLux sensors across a variety of applications in both natural systems, such as rivers and leachate into groundwater, and engineered systems, including drinking water and waste water treatment. Recent work has focused on standardising the fluorescence output across the UviLux range of sensors, reporting data in quinine sulphate units (QSU), which enables the output from two different fluorometers to be directly compared both to each other, and to bench-top data. A key advantage of deploying multiple sensors is the ability to fingerprint the fluorescence, by providing, for example, a Tryptophan/CDOM ratio. From the data collected, the ratio of the different fluorescence regions has been shown to provide more robust in situ data and help identify true temporal variations and patterns across multiple applications and sampling locations.

Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia.

PubMed

Idakieva, Krassimira; Siddiqui, Nurul I; Parvanova, Katja; Nikolov, Peter; Gielens, Constant

2006-04-01

The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.

[Three dimensional fluorescent characteristics of soil dissolved organic matter (DOM) in Jiaozhou Bay coastal wetlands, China].

PubMed

Zi, Yuan Yuan; Kong, Fan Long; Xi, Min; Li, Yue; Yang, Ling

2016-12-01

In order to elucidate the structure characteristics of soil dissolved organic matter (DOM) and analyze the sources in Jiaozhou Bay coastal wetlands, four typical types of wetlands around Jiaozhou Bay were chosen, including Spartina anglica wetland, the barren wetland, Suaeda glauca wetland and Phragmites australis wetland. The soil samples were collected in January 2014. The contents of soil DOM were determined and the spectral analysis was made by three-dimensional fluorescent technology. The results showed that the contents of soil dissolved organic carbon (DOC) in four types of wetlands all decreased with the increasing soil depth, and S. anglica wetland ranked the first in the contents of soil DOC, followed by the barren wetland, S. glauca wetland and P. australis wetland. Five fluorescence peaks including B, T, A, D and C were found in the three-dimensional fluorescence spectrum (3DEEMs), indicating tyrosine-like, tryptophan-like, phenol-like, soluble microbial byproduct-like and humic acid-like- substances, respectively. Fluorescence integration (FRI) was applied in the qualitative analysis of five components. The results showed that tryptophan-like, phenol-like and tyrosine-like substances ranked in top three in content, followed by soluble microbial byproduct-like and humic acid-like substances which were not significantly different. Pearson correlation analysis demonstrated that a positive correlation existed between any two of the five components of DOM, and they were all positively related to DOC content. In addition, there existed different correlations between the five components of DOM and total phosphorus (TP), available phosphorus (AP) and total nitrogen (TN). The soil DOM in the four types of wetlands was mainly produced by biotic interactions, and the degree of humification was relatively low.

Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microchip electrophoresis.

PubMed

Schulze, Philipp; Ludwig, Martin; Kohler, Frank; Belder, Detlev

2005-03-01

Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in microchip electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.

Interaction of albumin with perylene-diimides with aromatic substituents

NASA Astrophysics Data System (ADS)

Farooqi, Mohammed; Penick, Mark; Burch, Jessica; Negrete, George; Brancaleon, Lorenzo

2015-03-01

Polyaromatic hydrocarbons (PAH) binding to proteins remains one of the fundamental aspects of research in biophysics. Ligand binding can regulate the function of proteins. Binding to small ligands remains a very important aspect in the study of the function of many proteins. Perylene diimide or PDI derivatives have attracted initial interest as industrial dyes and pigments. Recently, much attention has been focused on their strong π - π stacks resulting from the large PDI aromatic core. These PDI stacks have distinct optical properties, and provide informative models that mimic the light-harvesting system and initial charge separation and charge transfer in the photosynthetic system. The absorption property of PDI derivatives may be largely tuned from visible to near-infrared region by chemical modifications at the bay-positions. We are currently studying a new class of PDI derivatives with substituents made of the side chains of aromatic amino acids (Tyrosine, Tryptophan and Phenylalanine). We have looked at the fluorescence absorption and emission of these PDIs in water and other organic solvents. PDIs show evidence of dimerization and possible aggregation. We also present binding studies of these PDIs with Human Serum Albumin (HSA). The binding was studied using fluorescence emission quenching of the HSA Tryptophan residue. Stern-Volmer equation is used to derive the quenching constants. PDI binding to HSA also has an effect on the fluorescence emission of the PDIs themselves by red shifting the spectra. Funded by RCMI grant.

Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding.

PubMed

Jacob, Maik H; D'Souza, Roy N; Schwarzlose, Thomas; Wang, Xiaojuan; Huang, Fang; Haas, Elisha; Nau, Werner M

2018-04-26

Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.

Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

PubMed Central

Cherepanov, A V; de Vries, S

2001-01-01

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity. PMID:11721015

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

Identifying changes in dissolved organic matter content and characteristics by fluorescence spectroscopy coupled with self-organizing map and classification and regression tree analysis during wastewater treatment.

PubMed

Yu, Huibin; Song, Yonghui; Liu, Ruixia; Pan, Hongwei; Xiang, Liancheng; Qian, Feng

2014-10-01

The stabilization of latent tracers of dissolved organic matter (DOM) of wastewater was analyzed by three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy coupled with self-organizing map and classification and regression tree analysis (CART) in wastewater treatment performance. DOM of water samples collected from primary sedimentation, anaerobic, anoxic, oxic and secondary sedimentation tanks in a large-scale wastewater treatment plant contained four fluorescence components: tryptophan-like (C1), tyrosine-like (C2), microbial humic-like (C3) and fulvic-like (C4) materials extracted by self-organizing map. These components showed good positive linear correlations with dissolved organic carbon of DOM. C1 and C2 were representative components in the wastewater, and they were removed to a higher extent than those of C3 and C4 in the treatment process. C2 was a latent parameter determined by CART to differentiate water samples of oxic and secondary sedimentation tanks from the successive treatment units, indirectly proving that most of tyrosine-like material was degraded by anaerobic microorganisms. C1 was an accurate parameter to comprehensively separate the samples of the five treatment units from each other, indirectly indicating that tryptophan-like material was decomposed by anaerobic and aerobic bacteria. EEM fluorescence spectroscopy in combination with self-organizing map and CART analysis can be a nondestructive effective method for characterizing structural component of DOM fractions and monitoring organic matter removal in wastewater treatment process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interaction of Chelerythrine with Keyhole Limpet Hemocyanin: a Fluorescence Spectroscopy and Molecular Docking Study

NASA Astrophysics Data System (ADS)

Zhong, M.; Long, R. Q.; Wang, Y. H.; Chen, C. L.

2018-05-01

The quenching mechanism between chelerythrine (CHE) and keyhole limpet hemocyanin (KLH) was investigated using fluorescence spectroscopy and molecular docking. The experiments were conducted at three different temperatures (293, 298, and 303 K). The results revealed that the intrinsic fluorescence of KLH was strongly quenched by CHE through a static quenching mechanism. The thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction were calculated, indicating that the interaction between CHE and KLH was spontaneous and that van der Waals forces and hydrogen bond formation played major roles in the binding process. The intrinsic fluorescence of the tyrosine and tryptophan residues in KLH was studied by synchronous fluorescence, which suggested that CHE changed the conformation of KLH. Finally, molecular docking was used to obtain detailed information on the binding sites and binding affinities between CHE and KLH.

Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor.

PubMed

Joshi, Bishnu Prasad; Park, Junwon; Lee, Wan In; Lee, Keun-Hyeung

2009-05-15

A novel fluorescent peptide sensor containing tryptophan (donor) and dansyl fluorophore (acceptor) was synthesized for monitoring heavy and transition metal (HTM) ions on the basis of metal ion binding motif (Cys-X-X-X-Cys). The peptide probe successfully exhibited a turn on and ratiometric response for several heavy metal ions such as Hg(2+), Cd(2+), Pb(2+), Zn(2+), and Ag(+) in aqueous solution. The enhancements of emission intensity were achieved in the presence of the HTM ions by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The detection limits of the sensor for Cd(2+), Pb(2+), Zn(2+), and Ag(+) were lower than the EPA's drinking water maximum contaminant levels (MCL). We described the fluorescent enhancement, binding affinity, and detection limit of the peptide probe for HTM ions.

Performance and membrane fouling of a step-fed submerged membrane sequencing batch reactor treating swine biogas digestion slurry.

PubMed

Han, Zhiying; Chen, Shixia; Lin, Xiaochang; Yu, Hongjun; Duan, Li'an; Ye, Zhangying; Jia, Yanbo; Zhu, Songming; Liu, Dezhao

2018-01-02

To identify the performance of step-fed submerged membrane sequencing batch reactor (SMSBR) treating swine biogas digestion slurry and to explore the correlation between microbial metabolites and membrane fouling within this novel reactor, a lab-scale step-fed SMSBR was operated under nitrogen loading rate of 0.026, 0.052 and 0.062 g NH 4 + -N (gVSS·d) -1 . Results show that the total removal efficiencies for NH 4 + -N, total nitrogen and chemical oxygen demand in the reactor (>94%, >89% and >97%, respectively) were high during the whole experiment. However, the cycle removal efficiency of NH 4 + -N decreased significantly when the nitrogen loading rate was increased to 0.062 g NH 4 + -N (gVSS·d) -1 . The total removal efficiency of total phosphorus in the step-fed SMSBR was generally higher than 75%, though large fluctuations were observed during the experiments. In addition, the concentrations of microbial metabolites, i.e., soluble microbial products (SMP) and extracellular polymeric substances (EPS) from activated sludge increased as nitrogen loading rate increased, both showing quadratic equation correlations with viscosity of the mixed liquid in the step-fed SMSBR (both R 2 > 0.90). EPS content was higher than SMP content, while protein (PN) was detected as the main component in both SMP and EPS. EPS PN was found to be well correlated with transmembrane pressure, membrane flux and the total membrane fouling resistance. Furthermore, the three-dimensional excitation-emission matrix fluorescence spectroscopy results suggested the tryptophan-like protein as one of the main contributors to the membrane fouling. Overall, this study showed that the step-fed SMSBR could be used to treat swine digestion slurry at nitrogen loading rate of 0.052 g NH 4 + -N (gVSS·d) -1 , and the control strategy of membrane fouling should be developed based on reducing the tryptophan-like PN in EPS.

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

PubMed Central

Chen, Qiang; Cheng, Mary Hongying; Xu, Yan; Tang, Pei

2010-01-01

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins. PMID:20858424

Tuning electronic transport via hepta-alanine peptides junction by tryptophan doping.

PubMed

Guo, Cunlan; Yu, Xi; Refaely-Abramson, Sivan; Sepunaru, Lior; Bendikov, Tatyana; Pecht, Israel; Kronik, Leeor; Vilan, Ayelet; Sheves, Mordechai; Cahen, David

2016-09-27

Charge migration for electron transfer via the polypeptide matrix of proteins is a key process in biological energy conversion and signaling systems. It is sensitive to the sequence of amino acids composing the protein and, therefore, offers a tool for chemical control of charge transport across biomaterial-based devices. We designed a series of linear oligoalanine peptides with a single tryptophan substitution that acts as a "dopant," introducing an energy level closer to the electrodes' Fermi level than that of the alanine homopeptide. We investigated the solid-state electron transport (ETp) across a self-assembled monolayer of these peptides between gold contacts. The single tryptophan "doping" markedly increased the conductance of the peptide chain, especially when its location in the sequence is close to the electrodes. Combining inelastic tunneling spectroscopy, UV photoelectron spectroscopy, electronic structure calculations by advanced density-functional theory, and dc current-voltage analysis, the role of tryptophan in ETp is rationalized by charge tunneling across a heterogeneous energy barrier, via electronic states of alanine and tryptophan, and by relatively efficient direct coupling of tryptophan to a Au electrode. These results reveal a controlled way of modulating the electrical properties of molecular junctions by tailor-made "building block" peptides.

Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

NASA Astrophysics Data System (ADS)

Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

2015-11-01

The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

Interaction mechanism between berberine and the enzyme lysozyme

NASA Astrophysics Data System (ADS)

Cheng, Ling-Li; Wang, Mei; Wu, Ming-Hong; Yao, Si-De; Jiao, Zheng; Wang, Shi-Long

2012-11-01

In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)•) react with Trp (K = 3.4 × 109 M-1 s-1) via electron transfer to give the radical cation (Trp/NH•+) and neutral radical of Trp (TrpN•). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR∗ and Trp or Lys were determined to be electron transfer process.

Role for tryptophan in regulation of protein synthesis in porcine muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, F.D.; Smith, T.K.; Bayley, H.S.

1988-04-01

Experiments were conducted to determine the effect of varying concentrations of dietary tryptophan on growth rate and protein synthesis in edible muscle tissues of growing swine. A total of 45 immature swine (initial weight approximately 24 kg) were fed corn-gelatin diets containing 0.5 (n = 8), 0.8 (n = 10), 1.3 (n = 10), 1.5 (n = 7) or 2.0 (n = 10) g tryptophan/kg diet for 35 d. Animals fed 0.5 and 0.8 g tryptophan/kg grew more slowly, consumed less feed and had a lower efficiency of feed utilization than animals fed higher concentrations of tryptophan. Thirty similar animalsmore » were used in a second experiment. Diets containing 0.5, 0.8, 1.0, 1.5 or 2.0 g tryptophan/kg diet (n = 6) were fed for 14 d, after which all animals were killed and samples were taken of longissimus dorsi, triceps brachii and biceps femoris. Protein synthetic activity was determined by monitoring the incorporation of (/sup 14/C)phenylalanine into protein in vitro. There was no significant difference in synthetic activity between different muscle types. There was no effect of diet on the activity of the muscle soluble protein fraction. The activity of the muscle ribosomal fraction, however, was positively correlated with increasing concentrations of dietary tryptophan. It was concluded that tryptophan has the potential to regulate muscle protein synthesis in a manner beyond serving simply as a component of protein.« less

Characterization of CDOM of river waters in China using fluorescence excitation-emission matrix and regional integration techniques

NASA Astrophysics Data System (ADS)

Zhao, Ying; Song, Kaishan; Shang, Yingxin; Shao, Tiantian; Wen, Zhidan; Lv, Lili

2017-08-01

The spatial characteristics of fluorescent dissolved organic matter (FDOM) components in river waters in China were first examined by excitation-emission matrix spectra and fluorescence regional integration (FRI) with the data collected during September to November between 2013 and 2015. One tyrosine-like (R1), one tryptophan-like (R2), one fulvic-like (R3), one microbial protein-like (R4), and one humic-like (R5) components have been identified by FRI method. Principal component analysis (PCA) was conducted to assess variations in the five FDOM components (FRί (ί = 1, 2, 3, 4, and 5)) and the humification index for all 194 river water samples. The average fluorescence intensities of the five fluorescent components and the total fluorescence intensities FSUM differed under spatial variation among the seven major river basins (Songhua, Liao, Hai, Yellow and Huai, Yangtze, Pearl, and Inflow Rivers) in China. When all the river water samples were pooled together, the fulvic-like FR3 and the humic-like FR5 showed a strong positive linear relationship (R2 = 0.90, n = 194), indicating that the two allochthonous FDOM components R3 and R5 may originate from similar sources. There is a moderate strong positive correlation between the tryptophan-like FR2 and the microbial protein-like FR4 (R2 = 0.71, n = 194), suggesting that parts of two autochthonous FDOM components R2 and R4 are likely from some common sources. However, the total allochthonous substance FR(3+5) and the total autochthonous substances FR(1+2+4) exhibited a weak correlation (R2 = 0.40, n = 194). Significant positive linear relationships between FR3 (R2 = 0.69, n = 194), FR5 (R2 = 0.79, n = 194), and chromophoric DOM (CDOM) absorption coefficient a(254) were observed, which demonstrated that the CDOM absorption was dominated by the allochthonous FDOM components R3 and R5.

Identification of a Potent Tryptophan-based TRPM8 Antagonist With in vivo Analgesic Activity.

PubMed

Bertamino, Alessia; Iraci, Nunzio; Ostacolo, Carmine; Ambrosino, Paolo; Musella, Simona; Di Sarno, Veronica; Ciaglia, Tania; Pepe, Giacomo; Sala, Marina; Soldovieri, Maria Virginia; Mosca, Ilaria; Gonzalez-Rodriguez, Sara; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; Novellino, Ettore; Taglialatela, Maurizio; Campiglia, Pietro; Gomez-Monterrey, Isabel M

2018-06-25

TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N,N'-dibenzyl tryptamine 1, a potent TRPM8 antagonist, were prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably-transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC 50 0.2±0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.

Multispectroscopic and bioimaging approach for the interaction of rhodamine 6G capped gold nanoparticles with bovine serum albumin.

PubMed

Manjubaashini, N; Kesavan, Mookkandi Palsamy; Rajesh, Jegathalaprathaban; Daniel Thangadurai, T

2018-06-01

Binding interaction of Bovine Serum Albumin (BSA) with newly prepared rhodamine 6G-capped gold nanoparticles (Rh6G-Au NPs) under physiological conditions (pH 7.2) was investigated by a wide range of photophysical techniques. Rh6G-Au NPs caused the static quenching of the intrinsic fluorescence of BSA that resulted from the formation of ground-state complex between BSA and Rh6G-Au NPs. The binding constant from fluorescence quenching method (K a  = 1.04 × 10 4  L mol -1 ; LoD = 14.0 μM) is in accordance with apparent association constant (K app  = 1.14 × 10 1  M -1 ), which is obtained from absorption spectral studies. Förster resonance energy transfer (FRET) efficiency between the tryptophan (Trp) residue of BSA and fluorophore of Rh6G-Au NPs during the interaction was calculated to be 90%. The free energy change (ΔG = -23.07 kJ/mol) of BSA-Rh6G-Au NPs complex was calculated based on modified Stern-Volmer Plot. The time-resolved fluorescence analysis confirmed that quenching of BSA follows static mechanism through the formation of ground state complex. Furthermore, synchronous and three-dimensional fluorescence measurement, Raman spectral analysis and Circular Dichroism spectrum results corroborate the strong binding between Rh6G-Au NPs and BSA, which causes the conformational changes on BSA molecule. In addition, fluorescence imaging experiments of BSA in living human breast cancer (HeLa) cells was successfully demonstrated, which articulated the value of Rh6G-Au NPs practical applications in biological systems. Copyright © 2018. Published by Elsevier B.V.

Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation.

PubMed

Hsiao, Paoyuan; Sanjaya; Su, Ruey-Chih; Teixeira da Silva, Jaime A; Chan, Ming-Tsair

2007-03-01

Gene transformation is an integral tool for plant genetic engineering. All antibiotic resistant genes currently employed are of bacterial origin and their presence in the field is undesirable. Therefore, we developed a novel and efficient plant native non-antibiotic selection system for the selection of transgenic plants in the model system Arabidopsis. This new system is based on the enhanced expression of Arabidopsis tryptophan synthase beta 1 (AtTSB1) and the use of 5-methyl-tryptophan (5MT, a tryptophan [Trp] analog) and/or CdCl2 as selection agent(s). We successfully integrated an expression cassette containing an AtT-SB1 cDNA driven by a cauliflower mosaic virus 35S promoter into Arabidopsis by floral dip transformation. Transgenic plants were efficiently selected on MS medium supplemented with 75 microM 5MT or 300 microM CdCl2 devoid of antibiotics. TSB1 selection was as efficient as the conventional hygromycin selection system. Northern blot analysis of transgenic plants selected by 5MT and CdCl2 revealed increased TSB1 mRNA transcript whereas uneven transcript levels of hygromycin phosphotransferase II (hpt) (control) was observed. Gas chromatography-mass spectrometry revealed 10-15 fold greater free Trp content in AtT-SB1 transgenic plants than in wild-type plants grown with or without 5MT or CdCl2. Taken together, the TSB1 system provides a novel selection system distinct from conventional antibiotic selection systems.

Effect of granular activated carbon addition on the effluent properties and fouling potentials of membrane-coupled expanded granular sludge bed process.

PubMed

Ding, An; Liang, Heng; Qu, Fangshu; Bai, Langming; Li, Guibai; Ngo, Huu Hao; Guo, Wenshan

2014-11-01

To mitigate membrane fouling of membrane-coupled anaerobic process, granular activated carbon (GAC: 50 g/L) was added into an expanded granular sludge bed (EGSB). A short-term ultrafiltration test was investigated for analyzing membrane fouling potential and underlying fouling mechanisms. The results showed that adding GAC into the EGSB not only improved the COD removal efficiency, but also alleviated membrane fouling efficiently because GAC could help to reduce soluble microbial products, polysaccharides and proteins by 26.8%, 27.8% and 24.7%, respectively, compared with the control system. Furthermore, excitation emission matrix (EEM) fluorescence spectroscopy analysis revealed that GAC addition mainly reduced tryptophan protein-like, aromatic protein-like and fulvic-like substances. In addition, the resistance distribution analysis demonstrated that adding GAC primarily decreased the cake layer resistance by 53.5%. The classic filtration mode analysis showed that cake filtration was the major fouling mechanism for membrane-coupled EGSB process regardless of the GAC addition. Copyright © 2014 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, F.D.; Smith, T.K.; Bayley, H.S.

Experiments were conducted to determine the effect of varying concentrations of dietary tryptophan on growth rate and protein synthesis in edible muscle tissues of growing swine. A total of 45 immature swine (initial weight approximately 24 kg) were fed corn-gelatin diets containing 0.5 (n = 8), 0.8 (n = 10), 1.3 (n = 10), 1.5 (n = 7) or 2.0 (n = 10) g tryptophan/kg diet for 35 d. Animals fed 0.5 and 0.8 g tryptophan/kg grew more slowly, consumed less feed and had a lower efficiency of feed utilization than animals fed higher concentrations of tryptophan. Thirty similar animalsmore » were used in a second experiment. Diets containing 0.5, 0.8, 1.0, 1.5 or 2.0 g tryptophan/kg diet (n = 6) were fed for 14 d, after which all animals were killed and samples were taken of longissimus dorsi, triceps brachii and biceps femoris. Protein synthetic activity was determined by monitoring the incorporation of (/sup 14/C)phenylalanine into protein in vitro. There was no significant difference in synthetic activity between different muscle types. There was no effect of diet on the activity of the muscle soluble protein fraction. The activity of the muscle ribosomal fraction, however, was positively correlated with increasing concentrations of dietary tryptophan. It was concluded that tryptophan has the potential to regulate muscle protein synthesis in a manner beyond serving simply as a component of protein.« less

Fluorescent spectral studies of the toxic effect of chlororganic pesticides on the biochemical parameters of synaptosomes

NASA Astrophysics Data System (ADS)

Giraev, K. M.; Bekshokov, K. S.; Ashurbekov, N. A.; Abdullaeva, N. M.; Israpov, E. Kh.; Gashimov, I. Sh.

2017-04-01

The results of the study of the fluorescence spectra of suspensions of synaptosomes, which have been exposed to a chlororganic pesticide, thiamethoxam, at a concentration of 50 MPC during different time periods, at the excitation/emission wavelengths of 290 ± 5/310-600, 340 ± 5/360-700, and 420 ± 5/450-800 nm are given for the first time. It has been demonstrated that the development of intoxication results in weakening of the fluorescence intensity of tryptophan, NAD(P)·H, derivatives of vitamin B6, and vitamin A and in an increase in the fluorescence of pyridoxic acid, lipofuscin, and flavin and porphyrin complexes. The results of the spectral studies indicate that the toxic effect of the chlororganic pesticide for the functioning of living systems is based on free radical toxicity.

Interaction of Human Hemoglobin with Methotrexate

NASA Astrophysics Data System (ADS)

Zaharia, M.; Gradinaru, R.

2015-05-01

This study focuses on the interaction between methotrexate and human hemoglobin using steady-state ultraviolet-visible and fluorescence quenching methods. Fluorescence quenching was found to be valuable in assessing drug binding to hemoglobin. The quenching of methotrexate is slightly smaller than the quenching observed with related analogs (dihydrofolate and tetrahydrofolate). The quenching studies were performed at four different temperatures and various pH values. The number of binding sites for tryptophan is ~1. Parameter-dependent assays revealed that electrostatic forces play an essential role in the methotrexate-hemoglobin interaction. Furthermore, the complex was easily eluted using gel filtration chromatography.

Two-photon excited fluorescence emission from hemoglobin

NASA Astrophysics Data System (ADS)

Sun, Qiqi; Zeng, Yan; Zhang, Wei; Zheng, Wei; Luo, Yi; Qu, Jianan Y.

2015-03-01

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.

PubMed

Alimova, Alexandra; Katz, A; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A; Zeylikovich, Roman; Alfano, R R

2007-01-01

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (<10), while for malignant specimens, the I(345)I(500) ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

Protein side chain rotational isomerization: A minimum perturbation mapping study

NASA Astrophysics Data System (ADS)

Haydock, Christopher

1993-05-01

A theory of the rotational isomerization of the indole side chain of tryptophan-47 of variant-3 scorpion neurotoxin is presented. The isomerization potential energy, entropic part of the isomerization free energy, isomer probabilities, transition state theory reaction rates, and indole order parameters are calculated from a minimum perturbation mapping over tryptophan-47 χ1×χ2 torsion space. A new method for calculating the fluorescence anisotropy from molecular dynamics simulations is proposed. The method is based on an expansion that separates transition dipole orientation from chromophore dynamics. The minimum perturbation potential energy map is inverted and applied as a bias potential for a 100 ns umbrella sampling simulation. The entropic part of the isomerization free energy as calculated by minimum perturbation mapping and umbrella sampling are in fairly close agreement. Throughout, the approximation is made that two glutamine and three tyrosine side chains neighboring tryptophan-47 are truncated at the Cβ atom. Comparison with the previous combination thermodynamic perturbation and umbrella sampling study suggests that this truncated neighbor side chain approximation leads to at least a qualitatively correct theory of tryptophan-47 rotational isomerization in the wild type variant-3 scorpion neurotoxin. Analysis of van der Waals interactions in a transition state region indicates that for the simulation of barrier crossing trajectories a linear combination of three specially defined dihedral angles will be superior to a simple side chain dihedral reaction coordinate.

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carra,J.; McHugh, C.; Mulligan, S.

2007-01-01

We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding tomore » a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.« less

Structural perturbation of proteins in low denaturant concentrations.

PubMed

Basak, S; Debnath, D; Haque, E; Ray, S; Chakrabarti, A

2001-01-01

The presence of very low concentrations of the widely used chemical denaturants, guanidinium chloride and urea, induce changes in the tertiary structure of proteins. We have presented results on such changes in four structurally unrelated proteins to show that such structural perturbations are common irrespective of their origin. Data representative of such structural changes are shown for the monomeric globular proteins such as horseradish peroxidase (HRP) from a plant, human serum albumin (HSA) and prothrombin from ovine blood serum, and for the membrane-associated, worm-like elongated protein, spectrin, from ovine erythrocytes. Structural alterations in these proteins were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of the denaturants, even at concentrations below 100 mM, were higher than those measured in absence of the denaturants. Both steady-state and time-resolved fluorescence emission properties of tryptophan and of the extrinsic probe PRODAN were used for monitoring conformational changes in the proteins in presence of different low concentrations of the denaturants. These results are consistent with earlier studies from our laboratory indicating structural perturbations in proteins at the tertiary level, keeping their native-like secondary structure and their biological activity more or less intact.

The stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

PubMed Central

2004-01-01

13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen–deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine at pH 7.8. 1H-NMR has also been used to follow the tryptophan-synthase-catalysed hydrogen–deuterium exchange of the α-protons of a range of L- and D-amino acids at pH 7.8. The pKa values of the α-protons of these amino acids have been estimated and we have determined whether or not their exchange rates can be predicted from their pKa values. With the exception of tryptophan and norleucine, the stereospecificities of the first-order α-proton exchange rates are independent of the size and electronegativity of the amino acid R-group. Similar results are obtained with the second-order α-proton exchange rates, except that both L-tryptophan and L-serine have much higher stereospecificities than all the other amino acids studied. PMID:15107013

Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase.

PubMed

Xu, Lisheng; Wang, Zhiyuan; Mao, Pingting; Liu, Junzhong; Zhang, Hongjuan; Liu, Qian; Jiao, Qing-Cai

2013-04-01

An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efficiently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40°C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a final S-phenyl-L-cysteine concentration of 38.6 g l(-1). Copyright © 2013 Elsevier Ltd. All rights reserved.

Sublethal doses of neonicotinoid imidacloprid can interact with honey bee chemosensory protein 1 (CSP1) and inhibit its function.

PubMed

Li, Hongliang; Tan, Jing; Song, Xinmi; Wu, Fan; Tang, Mingzhu; Hua, Qiyun; Zheng, Huoqing; Hu, Fuliang

2017-04-29

As a frequently used neonicotinoid insecticide, imidacloprid can impair the chemoreceptive behavior of honey bees even at sublethal doses, while the physiochemical mechanism has not been further revealed. Here, multiple fluorescence spectra, thermodynamic method, and molecular docking were used to study the interaction and the functional inhibition of imidacloprid to the recombinant CSP1 protein in Asian honey bee, Apis cerana. The results showed that the fluorescence intensity (λ em  = 332 nm) of CSP1 could be significantly quenched by imidacloprid in a dynamic mode. During the quenching process, ΔH > 0, ΔS > 0, indicating that the acting forces of imidacloprid with CSP1 are mainly hydrophobic interactions. Synchronous fluorescence showed that the fluorescence of CSP1 was mainly derived from tryptophan, and the hydrophobicity of tryptophan decreased with the increase of imidacloprid concentration. Molecular docking predicted the optimal pose and the amino acid composition of the binding process. Circular dichroism (CD) spectra showed that imidacloprid reduced the α-helix of CSP1 and caused the extension of the CSP1 peptide chain. In addition, the binding of CSP1 to floral scent β-ionone was inhibited by nearly 50% of the apparent association constant (K A ) in the presence of 0.28-2.53 ng/bee of imidacloprid, and the inhibition rate of nearly 95% at 3.75 ng/bee of imidacloprid at sublethal dose level. This study initially revealed the molecular physiochemical mechanism that sublethal doses of neonicotinoid still interact and inhibit the physiological function of the honey bees' chemoreceptive system. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluating the ameliorative effect of natural dissolved organic matter (DOM) quality on copper toxicity to Daphnia magna: improving the BLM.

PubMed

Al-Reasi, Hassan A; Smith, D Scott; Wood, Chris M

2012-03-01

Various quality predictors of seven different natural dissolved organic matter (DOM) and humic substances were evaluated for their influence on protection of Daphnia magna neonates against copper (Cu) toxicity. Protection was examined at 3 and 6 mg l(-1) of dissolved organic carbon (DOC) of each DOM isolate added to moderately hard, dechlorinated water. Other water chemistry parameters (pH, concentrations of DOC, calcium, magnesium and sodium) were kept relatively constant. Predictors included absorbance ratios Abs(254/365) (index of molecular weight) and Abs-octanol(254)/Abs-water(254) (index of lipophilicity), specific absorption coefficient (SAC(340); index of aromaticity), and fluorescence index (FI; index of source). In addition, the fluorescent components (humic-like, fulvic-like, tryptophan-like, and tyrosine-like) of the isolates were quantified by parallel factor analysis (PARAFAC). Up to 4-fold source-dependent differences in protection were observed amongst the different DOMs. Significant correlations in toxicity amelioration were found with Abs(254/365), Abs-octanol(254)/Abs-water(254), SAC(340), and with the humic-like fluorescent component. The relationships with FI were not significant and there were no relationships with the tryptophan-like or tyrosine-like fluorescent components at 3 mg C l(-1), whereas a negative correlation was seen with the fulvic-like component. In general, the results indicate that larger, optically dark, more lipophilic, more aromatic DOMs of terrigenous origin, with higher humic-like content, are more protective against Cu toxicity. A method for incorporating SAC(340) as a DOM quality indicator into the Biotic Ligand Model is presented; this may increase the accuracy for predicting Cu toxicity in natural waters.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction.

PubMed

Saha, Ranajay; Rakshit, Surajit; Pal, Samir Kumar

2013-11-01

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of C/N ratio on extracellular polymeric substances of activated sludge from an anoxic-aerobic sequencing batch reactor treating saline wastewater.

PubMed

Wang, Zichao; Gao, Mengchun; Xin, Yanjun; Ma, Dong; She, Zonglian; Wang, Zhe; Sun, Changqing; Ren, Yun

2014-01-01

The effect of C/N ratio on extracellular polymeric substances (EPS) of activated sludge was investigated in an anoxic-aerobic sequencing batch reactor (SBR) treating saline wastewater. The protein (PN) and protein/polysaccharide (PN/PS) ratio in the loosely bound EPS (LB-EPS) increased with the decrease of C/N ratio, whereas the PS in the LB-EPS decreased. The PS, PN and PN/PS ratio in the tightly bound EPS (TB-EPS) were independent of C/N ratio. Two fluorescence peaks in the LB-EPS and TB-EPS were identified at excitation/emission (Ex/Em) wavelengths of 275-280/335-340 nm and 220-225/330-340 nm by three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy, respectively. These peaks in LB-EPS and TB-EPS were, respectively, associated with tryptophan protein-like substances and aromatic protein-like substances. The tryptophan protein-like fluorescence peaks in LB-EPS showed blue shift along the Ex axis and red shift along the Em axis with the decrease of C/N ratio. Fourier transform infrared spectra suggested that the variation of C/N ratio had more distinct effect on the functional groups of protein in the LB-EPS than those in the TB-EPS. The sludge volume index value decreased with the increase of LB-EPS, but there was no correlation between SVI and TB-EPS.

The relationship of chromophoric dissolved organic matter parallel factor analysis fluorescence and polycyclic aromatic hydrocarbons in natural surface waters.

PubMed

Li, Sijia; Chen, Ya'nan; Zhang, Jiquan; Song, Kaishan; Mu, Guangyi; Sun, Caiyun; Ju, Hanyu; Ji, Meichen

2018-01-01

Polycyclic aromatic hydrocarbons (PAHs), a large group of persistent organic pollutants (POPs), have caused wide environmental pollution and ecological effects. Chromophoric dissolved organic matter (CDOM), which consists of complex compounds, was seen as a proxy of water quality. An attempt was made to understand the relationships of CDOM absorption parameters and parallel factor analysis (PARAFAC) components with PAHs under seasonal variation in the riverine, reservoir, and urban waters of the Yinma River watershed in 2016. These different types of water bodies provided wide CDOM and PAHs concentration ranges with CDOM absorption coefficients at a wavelength of 350 nm (a CDOM (350)) of 1.17-20.74 m -1 and total PAHs of 0-1829 ng/L. CDOM excitation-emission matrix (EEM) presented two fluorescent components, e.g., terrestrial humic-like (C1) and tryptophan-like (C2) were identified using PARAFAC. Tryptophan-like associated protein-like fluorescence often dominates the EEM signatures of sewage samples. Our finding is that seasonal CDOM EEM-PARAFAC and PAHs concentration showed consistent tendency indicated that PAHs were un-ignorable pollutants. However, the disparities in seasonal CDOM-PAH relationships relate to the similar sources of CDOM and PAHs, and the proportion of PAHs in CDOM. Overlooked and poorly appreciated, quantifying the relationship between CDOM and PAHs has important implications, because these results simplify ecological and health-based risk assessment of pollutants compared to the traditional chemical measurements.

Interactions of hemin with bovine serum albumin and human hemoglobin: A fluorescence quenching study

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2018-03-01

The binding interactions between hemin (Hmi) and bovine serum albumin (BSA) or human hemoglobin (HHb), respectively, have been examined in aqueous solution at pH = 7.4, applying UV-vis absorption, as well as steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative results received for both BSA and HHb intrinsic fluorescence proceeding from the interactions with hemin suggest the formation of stacking non-covalent and non-fluorescent complexes in both the Hmi-BSA and Hmi-HHb systems, with highly possible concurrent formation of a coordinate bond between a group on the protein surface and the metal in Hmi molecule. All the values of calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants point to the involvement of static quenching in both the systems studied. The blue shift in the synchronous fluorescence spectra imply the participation of both tryptophan and tyrosine residues in quenching of BSA and HHb intrinsic fluorescence. Depicted outcomes suggest that hemin is supposedly able to influence the physiological functions of BSA and HHb, the most important blood proteins, particularly in case of its overuse.

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

PubMed

Essar, D W; Eberly, L; Crawford, I P

1990-02-01

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.

The influence of indoxyl sulfate and ammonium on the autofluorescence of human urine.

PubMed

Perinchery, Sandeep Menon; Kuzhiumparambil, Unnikrishnan; Vemulpad, Subramanyam; Goldys, Ewa M

2010-01-15

Despite biological variability the spectral characteristics of undiluted human urine show relatively low autofluorescence at short UV (250-300nm) excitation. However with dilution the fluorescence intensity remarkably increases. This paper examines the mechanisms behind this effect, by using excitation-emission matrices. Corrections for the inner filter effect were made for improved understanding of the spectral patterns. We focused on three major fluorophores (tryptophan, indoxyl sulfate and 5-hydroxyindole-3-acetate) that are excited at these wavelengths, and whose content in urine is strongly linked with various health conditions. Their fluorescence was studied both individually and in combinations. We also examined the effect of ammonium on the fluorescence of these major fluorophores individually and in combinations. Through these studies we have identified the leading effects that reduce the UV fluorescence, namely higher concentration of indoxyl sulfate producing the inner filter effect and concentration quenching and quenching of fluorophores by ammonium. This result will assist in broader utilisation of UV fluorescence in medical diagnostics.

Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

NASA Astrophysics Data System (ADS)

Lewis, William; Williams, Maura; Franco, Walfre

2017-02-01

The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

pH-induced vesicle-to-micelle transition in amphiphilic diblock copolymer: investigation by energy transfer between in situ formed polymer embedded gold nanoparticles and fluorescent dye.

PubMed

Maiti, Chiranjit; Banerjee, Rakesh; Maiti, Saikat; Dhara, Dibakar

2015-01-01

The ability to regulate the formation of nanostructures through self-assembly of amphiphilic block copolymers is of immense significance in the field of biology and medicine. In this work, a new block copolymer synthesized by using reversible addition-fragmentation chain transfer (RAFT) polymerization technique from poly(ethylene glycol) monomethyl ether acrylate (PEGMA) and Boc-l-tryptophan acryloyloxyethyl ester (Boc-l-trp-HEA) was found to spontaneously form pH-responsive water-soluble nanostructures after removal of the Boc group. While polymer vesicles or polymerosomes were formed at physiological pH, the micelles were formed at acidic pH (< 5.2), and this facilitated a pH-induced reversible vesicle-to-micelle transition. Formation of these nanostructures was confirmed by different characterization techniques, viz. transmission electron microscopy, dynamic light scattering, and steady-state fluorescence measurements. Further, these vesicles were successfully utilized to reduce HAuCl4 and stabilize the resulting gold nanoparticles (AuNPs). These AuNPs, confined within the hydrophobic shell of the vesicles, could participate in energy transfer process with fluorescent dye molecules encapsulated in the core of the vesicles, thus forming a nanometal surface energy transfer (NSET) pair. Subsequently, following the efficiency of energy transfer between this pair, it was possible to monitor the process of transition from vesicles to micelles. Thus, in this work, we have successfully demonstrated that NSET can be used to follow the transition between nanostructures formed by amphiphilic block copolymers.

Responses of soluble microbial products and extracellular polymeric substances to the presence of toxic 2,6-dichlorophenol in aerobic granular sludge system.

PubMed

Li, Kai; Wei, Dong; Yan, Tao; Du, Bin; Wei, Qin

2016-12-01

The objective of this study was to evaluate the responses of soluble microbial products (SMP) and extracellular polymeric substances (EPS) to the presence of toxic 2,6-dichlorophenol (2,6-DCP) in aerobic granular sludge (AGS) system. Batch experiment showed that NH 4 + -N removal efficiency significantly decreased from 99.6% to 47.2% in the toxic 2,6-DCP of 20 mg/L. Moreover, the inhibition degrees of 2,6-DCP on (SOUR) H , [Formula: see text] and [Formula: see text] were 7.8%, 32.1% and 9.5%, respectively. The main components of SMP, including protein (PN) and polysaccharide (PS) increased from 2.3 ± 0.74 and 16.8 ± 0.12 mg/L to 66.4 ± 0.56 and 18.0 ± 0.19 mg/L in the presence of 2,6-DCP. Three-dimensional excitation-emission matrix (3D-EEM) spectroscopy identified tryptophan PN-like, humic acid-like and fulvic acid-like substances in the control SMP, and their fluorescence intensities increased after exposure to 2,6-DCP. Synchronous fluorescence spectra suggested that the fluorescence quenching between EPS and 2,6-DCP was a static quenching process. The obtained results could provide insightful information on the responses of microbial products to AGS in the presence of toxic chlorophenols. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure of the cyclic peptide [W8S]contryphan Vn: effect of the tryptophan/serine substitution on trans-cis proline isomerization.

PubMed

Nepravishta, Ridvan; Mandaliti, Walter; Melino, Sonia; Eliseo, Tommaso; Paci, Maurizio

2014-12-01

The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

PubMed Central

Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

2015-01-01

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

Synthesis and biological evaluation of ¹⁸F-labeled fluoropropyl tryptophan analogs as potential PET probes for tumor imaging.

PubMed

Chiotellis, Aristeidis; Mu, Linjing; Müller, Adrienne; Selivanova, Svetlana V; Keller, Claudia; Schibli, Roger; Krämer, Stefanie D; Ametamey, Simon M

2013-01-01

In the search for an efficient, fluorine-18 labeled amino acid based radiotracer for tumor imaging with positron emission tomography (PET), two new tryptophan analogs were synthesized and characterized in vitro and in vivo. Both are tryptophan alkyl-derivatives, namely 2-(3-[(18)F]fluoropropyl)-DL-tryptophan ([(18)F]2-FPTRP) and 5-(3-[(18)F]fluoro-propyl)-DL-tryptophan ([(18)F]5-FPTRP). Standard reference compounds and precursors were prepared by multi step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 29-34% decay corrected yields with radiochemical purity over 99%. In vitro cell uptake assays showed that both compounds are substrates for amino acid transport and enter small cell lung cancer cells (NCI-H69) most probably almost exclusively via large neutral amino acids transporter(s) (LAT). Small animal PET imaging with xenograft bearing mice revealed high tumor/background ratios for [(18)F]2-FPTRP comparable to the well established tyrosine analog O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET). Radiometabolite studies showed no evidence of involvement of a biotransformation step in tumor accumulation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Thermal and Denaturation Studies of the Time-Resolved Fluorescence Decay of Human Superoxide Dismutase

NASA Astrophysics Data System (ADS)

Silva, Norberto De Jesus

Previous studies have shown that time-resolved fluorescence decay of various single tryptophan proteins is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. The thermal dependence of the lifetime distributions is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.

Anthropogenic signature of sediment organic matter probed by UV-Visible and fluorescence spectroscopy and the association with heavy metal enrichment.

PubMed

He, Wei; Lee, Jong-Hyun; Hur, Jin

2016-05-01

Sediment organic matter (SOM) was extracted in an alkaline solution from 43 stream sediments in order to explore the anthropogenic signatures. The SOM spectroscopic characteristics including excitation-emission matrix (EEM)-parallel factor analysis (PARAFAC) were compared for five sampling site groups classified by the anthropogenic variables of land use, population density, the loadings of organics and nutrients, and metal enrichment. The conventional spectroscopic characteristics including specific UV absorbance, absorbance ratio, and humification index did not properly discriminate among the different cluster groups except in the case of metal enrichment. Of the four decomposed PARAFAC components, humic-like and tryptophan-like fluorescence responded negatively and positively, respectively, to increasing degrees of the anthropogenic variables except for land use. The anthropogenic enrichment of heavy metals was positively associated with the abundance of tryptophan-like component. In contrast, humic-like component, known to be mostly responsible for metal binding, exhibited a decreasing trend corresponding with metal enrichment. These conflicting trends can be attributed to the overwhelmed effects of the coupled discharges of heavy metals and organic pollutants into sediments. Our study suggests that the PARAFAC components can be used as functional signatures to probe the anthropogenic influences on sediments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

PubMed Central

Ramírez-Sarmiento, César A.; Baez, Mauricio; Wilson, Christian A.M.; Babul, Jorge; Komives, Elizabeth A.; Guixé, Victoria

2013-01-01

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. PMID:23708365

Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.

PubMed

Ramírez-Sarmiento, César A; Baez, Mauricio; Wilson, Christian A M; Babul, Jorge; Komives, Elizabeth A; Guixé, Victoria

2013-05-21

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.

PubMed

Kleinschmidt, J H; Tamm, L K

1999-04-20

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.

A near-infrared BSA coated DNA-AgNCs for cellular imaging.

PubMed

Mu, Wei-Yu; Yang, Rui; Robertson, Akrofi; Chen, Qiu-Yun

2018-02-01

Near-infrared silver nanoclusters, have potential applications in the field of biosensing and biological imaging. However, less stability of most DNA-AgNCs limits their application. To obtain stable near-infrared fluorescence DNA-AgNCs for biological imaging, a new kind of near-infrared fluorescent DNA-Ag nanoclusters was constructed using the C3A rich aptamer as a synthesis template, GAG as the enhancer. In particular, a new DNA-AgNCs-Trp@BSA was obtained based on the self-assembly of bovine serum albumin (BSA) and tryptophan loaded DNA-AgNCs by hydrophobic interaction. This self-assembly method can be used to stabilize DNAn-Ag (n = 1-3) nanoclusters. Hence, the near-infrared fluorescence DNA-AgNCs-Trp@BSA was applied in cellular imaging of HepG-2 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Interactions of hemin with bovine serum albumin and human hemoglobin: A fluorescence quenching study.

PubMed

Makarska-Bialokoz, Magdalena

2018-03-15

The binding interactions between hemin (Hmi) and bovine serum albumin (BSA) or human hemoglobin (HHb), respectively, have been examined in aqueous solution at pH=7.4, applying UV-vis absorption, as well as steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative results received for both BSA and HHb intrinsic fluorescence proceeding from the interactions with hemin suggest the formation of stacking non-covalent and non-fluorescent complexes in both the Hmi-BSA and Hmi-HHb systems, with highly possible concurrent formation of a coordinate bond between a group on the protein surface and the metal in Hmi molecule. All the values of calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants point to the involvement of static quenching in both the systems studied. The blue shift in the synchronous fluorescence spectra imply the participation of both tryptophan and tyrosine residues in quenching of BSA and HHb intrinsic fluorescence. Depicted outcomes suggest that hemin is supposedly able to influence the physiological functions of BSA and HHb, the most important blood proteins, particularly in case of its overuse. Copyright © 2017 Elsevier B.V. All rights reserved.

Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

2016-04-01

The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

Improvement of activity and stability of chloroperoxidase by chemical modification

PubMed Central

Liu, Jian-Zhong; Wang, Min

2007-01-01

Background Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes. Results In order to improve the activity and stability of chloroperoxidase, chloroperoxidase was modified by citraconic anhydride, maleic anhydride or phthalic anhydride. The catalytic activities, thermostabilities and organic solvent tolerances of native and modified enzymes were compared. In aqueous buffer, modified chloroperoxidases showed similar Km values and greater catalytic efficiencies kcat/Km for both sulfoxidation and oxidation of phenol compared to native chloroperoxidase. Of these modified chloroperoxidases, citraconic anhydride-modified chloroperoxidase showed the greatest catalytic efficiency in aqueous buffer. These modifications of chloroperoxidase increased their catalytic efficiencies for sulfoxidation by 12%~26% and catalytic efficiencies for phenol oxidation by 7%~53% in aqueous buffer. However, in organic solvent (DMF), modified chloroperoxidases had lower Km values and higher catalytic efficiencies kcat/Km than native chloroperoxidase. These modifications also improved their thermostabilities by 1~2-fold and solvent tolerances of DMF. CD studies show that these modifications did not change the secondary structure of chloroperoxidase. Fluorescence spectra proved that these modifications changed the environment of tryptophan. Conclusion Chemical modification of epsilon-amino groups of lysine residues of chloroperoxidase using citraconic anhydride, maleic anhydride or phthalic anhydride is a simple and powerful method to enhance catalytic properties of enzyme. The improvements of the activity and stability of chloroperoxidase are related to side chain reorientations of aromatics upon both modifications. PMID:17511866

Sucralose Destabilization of Protein Structure.

PubMed

Chen, Lee; Shukla, Nimesh; Cho, Inha; Cohn, Erin; Taylor, Erika A; Othon, Christina M

2015-04-16

Sucralose is a commonly employed artificial sweetener that behaves very differently than its natural disaccharide counterpart, sucrose, in terms of its interaction with biomolecules. The presence of sucralose in solution is found to destabilize the native structure of two model protein systems: the globular protein bovine serum albumin and an enzyme staphylococcal nuclease. The melting temperature of these proteins decreases as a linear function of sucralose concentration. We correlate this destabilization to the increased polarity of the molecule. The strongly polar nature is manifested as a large dielectric friction exerted on the excited-state rotational diffusion of tryptophan using time-resolved fluorescence anisotropy. Tryptophan exhibits rotational diffusion proportional to the measured bulk viscosity for sucrose solutions over a wide range of concentrations, consistent with a Stokes-Einstein model. For sucralose solutions, however, the diffusion is dependent on the concentration, strongly diverging from the viscosity predictions, and results in heterogeneous rotational diffusion.

Calcium and organic matter removal by carbonation process with waste incineration flue gas towards improvement of leachate biotreatment performance.

PubMed

Zhang, Cheng; Zhu, Xuedong; Wu, Liang; Li, Qingtao; Liu, Jianyong; Qian, Guangren

2017-09-01

Municipal solid wastes incineration (MSWI) flue gas was employed as the carbon source for in-situ calcium removal from MSWI leachate. Calcium removal efficiency was 95-97% with pH of 10.0-11.0 over 100min of flue gas aeration, with both bound Ca and free Ca being removed effectively. The fluorescence intensity of tryptophan, protein-like and humic acid-like compounds increased after carbonation process. The decrease of bound Ca with the increase of precipitate indicated that calcium was mainly converted to calcium carbonate precipitate. It suggested that the interaction between dissolved organic matter and Ca 2+ was weakened. Moreover, 10-16% of chemical oxygen demand removal and the decrease of ultraviolet absorption at 254nm indicated that some organics, especially aromatic compound decreased via adsorption onto the surface of calcium carbonate. The results indicate that introduce of waste incineration flue gas could be a feasible way for calcium removal from leachate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultraviolet light emitting diodes and bio-aerosol sensing

NASA Astrophysics Data System (ADS)

Davitt, Kristina M.

Recent interest in compact ultraviolet (UV) light emitters has produced advances in material quality and device performance from aluminum-rich alloys of the nitride semiconductor system. The epitaxial growth of device structures from this material poses remarkable challenges, and state-of-the-art in semiconductor UV light sources at wavelengths shorter than 350 nm is currently limited to LEDs. A portion of the work presented in this thesis involves the design and characterization of UV LED structures, with particular focus on sub-300 nm LEDs which have only been demonstrated within the last four years. Emphasis has been placed on the integration of early devices with modest efficiencies and output powers into a practical, fluorescence-based bio-sensing instrument. The quality of AlGaInN and AlGaN-based materials is characterized by way of the performance of 340 nm and 290 nm LEDs respectively. A competitive level of device operation is achieved, although much room remains for improvement in the efficiency of light emission from this material system. A preliminary investigation of 300 nm LEDs grown on bulk AIN shows promising electrical and optical characteristics, and illustrates the numerous advantages that this native substrate offers to the epitaxy of wide bandgap nitride semiconductors. The application of UV LEDs to the field of bio-aerosol sensing is pursued by constructing an on-the-fly fluorescence detection system. A linear array of UV LEDs is designed and implemented, and the capability of test devices to excite native fluorescence from bacterial spores is established. In order to fully capitalize on the reduction in size afforded by LEDs, effort is invested in re-engineering the remaining sensor components. Operation of a prototype system for physically sorting bio-aerosols based on fluorescence spectra acquired in real-time from single airborne particles excited by a UV-LED array is demonstrated using the bio-fluorophores NADH and tryptophan. Sensor performance is shown to be ultimately linked to the material quality of high aluminum fraction nitrides, and is expected to show progress as this field matures.

Dendritic biomimicry: microenvironmental hydrogen-bonding effects on tryptophan fluorescence.

PubMed

Koenig, S; Müller, L; Smith, D K

2001-03-02

Two series of dendritically modified tryptophan derivatives have been synthesised and their emission spectra measured in a range of different solvents. This paper presents the syntheses of these novel dendritic structures and discusses their emission spectra in terms of both solvent and dendritic effects. In the first series of dendrimers, the NH group of the indole ring is available for hydrogen bonding, whilst in the second series, the indole NH group has been converted to NMe. Direct comparison of the emission wavelengths of analogous NH and NMe derivatives indicates the importance of the Kamlet-Taft solvent beta3 parameter, which reflects the ability of the solvent to accept a hydrogen bond from the NH group, an effect not possible for the NMe series of dendrimers. For the NH dendrimers, the attachment of a dendritic shell to the tryptophan subunit leads to a red shift in emission wavelength. This dendritic effect only operates in non-hydrogen-bonding solvents. For the NMe dendrimers, however, the attachment of a dendritic shell has no effect on the emission spectra of the indole ring. This proves the importance of hydrogen bonding between the branched shell and the indole NH group in causing the dendritic effect. This is the first time a dendritic effect has been unambiguously assigned to individual hydrogen-bonding interactions and indicates that such intramolecular interactions are important in dendrimers, just as they are in proteins. Furthermore, this paper sheds light on the use of tryptophan residues as a probe of the microenvironment within proteins--in particular, it stresses the importance of hydrogen bonds formed by the indole NH group.

Trimodal spectra for high discrimination of benign and malignant prostate tissue

NASA Astrophysics Data System (ADS)

Al Salhi, Mohamad; Masilamani, Vadivel; Trinka, Vijmasi; Rabah, Danny; Al Turki, Mohammed R.

2011-02-01

High false positives and over diagnosis is a major problem with management of prostate cancer. A non-invasive or a minimally invasive technique to accurately distinguish malignant prostate cancers from benign tumors will be extremely helpful to overcome this problem. In this paper, we had used three different fluorescence spectroscopy techniques viz., Fluorescence Emission Spectrum (FES), Stokes' Shift Spectrum (SSS) and Reflectance Spectrum (RS) to discriminate benign prostate tumor tissues (N=12) and malignant prostate cancer tissues (N=8). These fluorescence techniques were used to determine the relative concentration of naturally occurring biomolecules such as tryptophan, elastin, NADH and flavin which are found to be out of proportion in cancer tissues. Our studies show that combining all three techniques, benign and malignant prostate tissues could be classified with accuracy greater than 90%. This preliminary report is based on in vitro spectroscopy analysis. However, by employing fluorescence endoscopy techniques, this can be extended to in vivo analysis as well. This technique has the potential to identify malignant prostate tissues without surgery.

A new front-face optical cell for measuring weak fluorescent emissions with time resolution in the picosecond time scale.

PubMed

Gryczynski, Z; Bucci, E

1993-11-01

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Spectrometric studies on the interaction of fluoroquinolones and bovine serum albumin

NASA Astrophysics Data System (ADS)

Ni, Yongnian; Su, Shaojing; Kokot, Serge

2010-02-01

The interaction between fluoroquinolones (FQs), ofloxacin and enrofloxacin, and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis spectroscopy. It was demonstrated that the fluorescence quenching of BSA by FQ is a result of the formation of the FQ-BSA complex stabilized, in the main, by hydrogen bonds and van der Waals forces. The Stern-Volmer quenching constant, KSV, and the corresponding thermodynamic parameters, Δ H, Δ S and Δ G, were estimated. The distance, r, between the donor, BSA, and the acceptor, FQ, was estimated from fluorescence resonance energy transfer (FRET). The effect of FQ on the conformation of BSA was analyzed with the aid of UV-vis absorbance spectra and synchronous fluorescence spectroscopy. Spectral analysis showed that the two FQs affected the conformation of the BSA but in a different manner. Thus, with ofloxacin, the polarity around the tryptophan residues decreased and the hydrophobicity increased, while for enrofloxacin, the opposite effect was observed.

Probing the binding of phenolic aldehyde vanillin with bovine serum albumin: Evidence from spectroscopic and docking approach.

PubMed

Siddiqui, Gufran Ahmed; Siddiqi, Mohammad Khursheed; Khan, Rizwan Hasan; Naeem, Aabgeena

2018-05-08

The interactions of bovine serum albumin (BSA) with vanillin (VAN) were studied using UV-vis absorption, fluorescence, synchronous fluorescence, three dimensional fluorescence spectroscopy (3D), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and molecular docking techniques. The results revealed that VAN causes the static quenching of BSA by forming BSA-VAN complex. The thermodynamic parameters obtained using isothermal titration calorimetry (ITC) showed that the interaction between BSA and VAN is spontaneous and hydrogen bonding, van der Waals forces are mainly involved in stabilizing the complex. The distance between the donor and the acceptor was analyzed using fluorescence resonance energy transfer (FRET) which showed Forster distance of 2.58 nm. Molecular docking technique was applied to study the modes of interaction between BSA-VAN system and it was found that VAN bound to the sub-domain IIA of BSA. Structural analysis using 3D, synchronous fluorescence FTIR, and CD showed that upon binding of VAN, BSA exhibits small micro-environmental changes around tryptophan amino acid residue. Copyright © 2018. Published by Elsevier B.V.

Comprehensive study of interaction between biocompatible PEG-InP/ZnS QDs and bovine serum albumin.

PubMed

Sannaikar, M S; Inamdar, Laxmi S; Pujar, G H; Wari, M N; Balasinor, Nafisa H; Inamdar, S R

2018-05-01

Polyethylene glycol (PEG) surface modified biocompatible InP/ZnS quantum dots (QDs) act as a potential alternative for conventional carcinogenic cadmium-based quantum dots for in vivo and in vitro studies. Comprehensively, we studied the interaction between a model protein bovine serum albumin (BSA) and PEGylated toxic free InP/ZnS QDs using various spectroscopic tools such as absorption, fluorescence quenching, time resolved and synchronous fluorescence spectroscopic measurements. These studies principally show that tryptophan (Trp) residues of BSA have preferable binding affinity towards PEG-InP/ZnS QDs surface and a blue shift in Trp fluorescence emission is a signature of conformational changes in its hydrophobic microenvironment. Photoluminescence (PL) intensity of Trp is quenched by ground state complex formation (static quenching) at room temperature. However, InP/ZnS@BSA conjugates become unstable with increasing temperature and PL intensity of Trp is quenched via dynamic quenching by PEG-InP/ZnS QDs. Experimentally determined thermodynamic parameters for these conjugates have shown spontaneity, entropy driven and exothermic nature of bio-conjugation. The calculated binding affinity (n ≅ 1, Hill coefficient) suggest that the affinity of InP/ZnS QDs for a BSA protein is not dependent on whether or not other BSA proteins are already bound to the QD surface. Energy transfer efficiency (E), Trp residue to InP/ZnS QDs distances and energy transfer rate (k T ) were all obtained from FÖrster resonance energy. Copyright © 2017 John Wiley & Sons, Ltd.

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.

PubMed

Sannigrahi, Achinta; Maity, Pabitra; Karmakar, Sanat; Chattopadhyay, Krishnananda

2017-03-02

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

PubMed

Hussain, Shina; Andrews, Diann; Hill, Bruce C

2017-12-05

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., T M ) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the T M by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the T M by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the T M of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

Optical properties and antimicrobial effects of silver nanoparticles synthesized by femtosecond laser photoreduction

NASA Astrophysics Data System (ADS)

dos Santos Courrol, Daniella; Regina Borges Lopes, Carla; da Silva Cordeiro, Thiago; Regina Franzolin, Marcia; Dias Vieira Junior, Nilson; Elgul Samad, Ricardo; Coronato Courrol, Lilia

2018-07-01

Silver nanoparticles exhibit a powerful antimicrobial action showing a pronounced potential to be widely used against drug resistance bacteria. The present work describes the optical properties and antimicrobial effect of silver nanoparticles produced by femtosecond laser photoreduction of AgNO3 in the presence of tryptophan water solution. The advantages of this method are the absence of hazardous chemical reducing agents in the solution, and the versatile dimensional control achieved. The synthesized silver nanoparticles were characterized by absorption and fluorescence spectroscopy and their antibacterial activity were determined by monitoring the cell viability of Escherichia coli. The effects of the silver nanoparticles concentration and laser parameters (exposure time and pulse energy), on the formation of the nanoparticles, and its influence on the bacteria growth inhibition were studied. The prepared silver nanoparticles exhibited suitable antimicrobial properties. The results demonstrated that the nanoparticles concentration plays an important role in their bactericidal efficacy. The increase in the laser energy caused an increase in E. coli growth inhibition. Irradiations with energies around 300 μJ for 60 min presented high antimicrobial activity due to the presence of kynurenine, sub product of tryptophan photolysis. The first-time formation mechanism of tryptophan silver nanoparticles in high optical intensities was also discussed.

The contribution of component variation and phytoplankton growth to the distribution variation of chromophoric dissolved organic matter content in a mid-latitude subtropical drinking water source reservoir for two different seasons.

PubMed

Sun, Qiyuan; Jiang, Juan; Zheng, Yuyi; Wang, Feifeng; Wu, Chunshan; Xie, Rong-Rong

2017-07-01

The distribution variation in chromophoric dissolved organic matter (CDOM) content in mid-latitude subtropical drinking water source reservoirs (MDWSRs) has great significance in the security of aquatic environments and human health. CDOM distribution is heavily influenced by biogeochemical processes and anthropogenic activity. However, little is known regarding the impact of component variation and phytoplankton growth on CDOM distribution variation in MDWSR. Therefore, samples were collected from a representative MDWSR (the Shanzai Reservoir) for analysis. CDOM absorption and fluorescence coupling with parallel factor analysis were measured and calculated. The results indicated that only two CDOM components were found in the surface water of Shanzai Reservoir, fulvic acid, and high-excitation tryptophan, originating from terrestrial and autochthonous sources, respectively. The types of components did not change with the season. The average molecular weight of CDOM increased in proportion to its fulvic acid content. The distribution variation in CDOM content mainly resulted from the variation in two CDOM components in summer and from high-excitation tryptophan in winter. Phytoplankton growth strongly influenced the distribution variation of CDOM content in summer; the metabolic processes of Cyanobacteria and Bacillariophyta consumed fulvic acid, while that of Cryptophyta produced high-excitation tryptophan.

Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima.

PubMed

Boville, Christina E; Romney, David K; Almhjell, Patrick J; Sieben, Michaela; Arnold, Frances H

2018-04-27

The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75 °C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability ( T 50 = 93 °C), its temperature profile is shifted such that high reactivity is observed at ∼37 °C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogues.

Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

NASA Astrophysics Data System (ADS)

Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

2017-06-01

We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

BSA binding and antimicrobial studies of branched polyethyleneimine-copper(II)bipyridine/phenanthroline complexes

NASA Astrophysics Data System (ADS)

Vignesh, Gopalaswamy; Arunachalam, Sankaralingam; Vignesh, Sivanandham; James, Rathinam Arthur

2012-10-01

The interaction of two water soluble branched polyethyleneimine-copper(II) complexes containing bipyridine/phenanthroline with bovine serum albumin (BSA) was studied by, UV-Visible absorption, fluorescence, lifetime measurements and circular dichroism spectroscopic techniques. The polymer-copper(II) complexes strongly quench the intrinsic fluorescence of BSA is the static quenching mechanism through hydrogen bonds and van der Waal's attraction. The distance r, between the BSA and the complexes seems to be less than 2 nm indicating that the energy transfer between the donor and acceptor occurs with high probability. Synchronous fluorescence studies indicate the binding of polymer-copper(II) complexes with BSA mostly changes the polarity around tryptophan residues rather than tyrosine residues. The circular dichroism studies indicate that the binding has induced considerable amount of conformational changes in the protein. The complexes also show some antibacterial and antifungal properties.

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II.

PubMed

Ranjbar, Samira; Ghobadi, Sirous; Khodarahmi, Reza; Nemati, Houshang

2012-05-01

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding. Copyright © 2012 Elsevier B.V. All rights reserved.

Are sanitation interventions a threat to drinking water supplies in rural India? An application of tryptophan-like fluorescence.

PubMed

Sorensen, J P R; Sadhu, A; Sampath, G; Sugden, S; Dutta Gupta, S; Lapworth, D J; Marchant, B P; Pedley, S

2016-01-01

Open defecation is practised by over 600 million people in India and there is a strong political drive to eliminate this through the provision of on-site sanitation in rural areas. However, there are concerns that the subsequent leaching of excreta from subsurface storage could be adversely impacting underlying groundwater resources upon which rural populations are almost completely dependent for domestic water supply. We investigated this link in four villages undergoing sanitary interventions in Bihar State, India. A total of 150 supplies were sampled for thermotolerant (faecal) coliforms (TTC) and tryptophan-like fluorescence (TLF): an emerging real-time indicator of faecal contamination. Sanitary risk inspections were also performed at all sites, including whether a supply was located within 10 m of a toilet, the recommended minimum separation. Overall, 18% of water supplies contained TTCs, 91% of which were located within 10 m of a toilet, 58% had TLF above detection limit, and sanitary risk scores were high. Statistical analysis demonstrated TLF was an effective indicator of TTC presence-absence, with a possibility of TTCs only where TLF exceeded 0.4 μg/L dissolved tryptophan. Analysis also indicated proximity to a toilet was the only significant sanitary risk factor predicting TTC presence-absence and the most significant predictor of TLF. Faecal contamination was considered a result of individual water supply vulnerability rather than indicative of widespread leaching into the aquifer. Therefore, increasing faecal contamination of groundwater-derived potable supplies is inevitable across the country as uptake of on-site sanitation intensifies. Communities need to be aware of this link and implement suitable decentralised low-cost treatment of water prior to consumption and improve the construction and protection of new supplies. Copyright © 2015 British Geological Survey, NERC. Published by Elsevier Ltd.. All rights reserved.

Coffee extracts suppress tryptophan breakdown in mitogen-stimulated peripheral blood mononuclear cells.

PubMed

Gostner, Johanna M; Schroecksnadel, Sebastian; Jenny, Marcel; Klein, Angela; Ueberall, Florian; Schennach, Harald; Fuchs, Dietmar

2015-01-01

Coffee consumption is considered to exert an influence on mood, the immune system, cardiovascular disease, and cancer development, but the mechanisms of action of coffee and its compounds are only partly known and understood. Immunomodulatory effects of filtered extracts of coffee and decaffeinated coffee as well as coffee compounds were investigated in human peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA). The activation of PBMCs was monitored by the breakdown of tryptophan to kynurenine via enzyme indoleamine 2,3-dioxygenase (IDO) and the production of the immune activation marker neopterin by GTP-cyclohydrolase I (GCH1). Both of these biochemical pathways are induced during cellular immune activation in response to the Th1-type cytokine interferon-γ (IFN-γ). Filtered extracts of coffee and decaffeinated coffee both suppressed tryptophan breakdown and neopterin formation in mitogen-stimulated PBMCs efficiently and in a dose-dependent manner. Of 4 coffee compounds tested individually, only gallic acid and less strong also caffeic acid had a consistent suppressive influence but also affected cell viability, whereas pure caffeine and chlorogenic acid exerted no relevant effect in the PBMC assay. The parallel influence of extracts on tryptophan breakdown and neopterin production shows an anti-inflammatory and immunosuppressive property of coffee extracts and some of its compounds. When extrapolating the in vitro results to in vivo, IFN-γ-mediated breakdown of tryptophan could be counteracted by the consumption of coffee or decaffeinated coffee. This may increase tryptophan availability for the biosynthesis of the neurotransmitter 5-hydroxytryptamine (serotonin) and thereby improve mood and quality of life.

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis.

PubMed

Williamson, Heather R; Sehanobish, Esha; Shiller, Alan M; Sanchez-Amat, Antonio; Davidson, Victor L

2017-02-21

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

Photochemical behavior of dissolved and colloidal organic matter in estuarine and oceanic waters.

PubMed

Zhu, Wen-Zhuo; Yang, Gui-Peng; Zhang, Hong-Hai

2017-12-31

Chromophoric dissolved organic matter (CDOM), carbohydrates, and amino acids were analyzed to investigate the photochemistry of total dissolved (<0.22μm) organic matter (DOM), high-molecular-weight (HMW, 1kDa-0.22μm) DOM and low-molecular-weight (LMW, <1kDa) DOM at stations in the Yangtze River and its coastal area, and in the Western Pacific Ocean. Results revealed that the humic-like and tryptophan-like CDOM fluorescent components in riverine, coastal, and oceanic surface waters were photodegraded during irradiation. However, the photochemical behavior of tyrosine-like component was obscured by the excessive fluorescence intensities of humic- and tryptophan-like fluorescent components. Light sensitivity varied depending on the source material; terrestrially derived DOM was more susceptible to irradiation than autochthonous DOM. In contrast to the expected photodegradation of CDOM, photo-induced synthetic reaction transformed the LMW matters to polysaccharides (PCHO) and degradation reaction decomposed the HMW DOM to Monosaccharides. Colloidal DOM preferentially underwent photodegradation, whereas permeate DOM mainly photosynthesized PCHO. The total hydrolysable amino acid (THAA) pool changed because of the additional input by the photodegradation of DOM or THAA itself. The compositions of THAA changed during the irradiation experiments, indicating that the different photochemical behavior of individual amino acids were related to their different original photoreactivities; the relatively stable amino acids (e.g., Ser and Gly) significantly accumulated during irradiation, whereas photo-active aromatic amino acids (e.g. Tyr and His) were prone to photodegradation. The data presented here demonstrated that irradiation significantly influence the conversion between dissolved and colloid organic matter. These results can promote the understanding of irradiation effect on the carbon and nitrogen cycle in riverine, estuarine and oceanic ecosystems. Copyright © 2017 Elsevier B.V. All rights reserved.

Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

PubMed

Wang, Jing-Zhang; Liu, Ji; Lin, Tao; Han, Yong-Guang; Luo, Yue; Xi, Lei; Du, Lin-Fang

2013-09-01

The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in β-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD. Copyright © 2013. Published by Elsevier Inc.

Conformational Dynamics of Titin PEVK Explored with FRET Spectroscopy

PubMed Central

Huber, Tamás; Grama, László; Hetényi, Csaba; Schay, Gusztáv; Fülöp, Lívia; Penke, Botond; Kellermayer, Miklós S.Z.

2012-01-01

The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics. PMID:23062340

Unveiling the water-associated conformational mobility in the active site of ascorbate peroxidase.

PubMed

Chao, Wei-Chih; Lin, Li-Ju; Lu, Jyh-Feng; Wang, Jinn-Shyan; Lin, Tzu-Chieh; Chen, Yi-Han; Chen, Yi-Ting; Yang, Hsiao-Ching; Chou, Pi-Tai

2018-03-01

We carried out comprehensive spectroscopic studies of wild type and mutants of ascorbate peroxidase (APX) to gain understanding of the conformational mobility of the active site. In this approach, three unnatural tryptophans were applied to replace the distal tryptophan (W41) in an aim to probe polarity/water environment near the edge of the heme-containing active site. 7-azatryptophan ((7-aza)Trp) is sensitive to environment polarity, while 2,7-azatryptophan ((2,7-aza)Trp) and 2,6-diazatryptophan ((2,6-aza)Trp) undergo excited-state water-catalyzed double and triple proton transfer, respectively, and are sensitive to the water network. The combination of their absorption, emission bands and the associated relaxation dynamics of these fluorescence probes, together with the Soret-band difference absorption and resonance Raman spectroscopy, lead us to unveil the water associated conformational mobility in the active site of APX. The results are suggestive of the existence of equilibrium between two different environments surrounding W41 in APX, i.e., the water-rich and water-scant forms with distinct fluorescence relaxation. Our results thus demonstrate for the first time the power of integrating multiple sensors (7-aza)Trp, (2,7-aza)Trp and (2,6-aza)Trp in probing the water environment of a specifically targeted Trp in proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes.

PubMed

Verly, Rodrigo M; Rodrigues, Magali A; Daghastanli, Katia Regina P; Denadai, Angelo Márcio L; Cuccovia, Iolanda M; Bloch, Carlos; Frézard, Frédéric; Santoro, Marcelo M; Piló-Veloso, Dorila; Bemquerer, Marcelo P

2008-01-01

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells.

PubMed

Pickhardt, Marcus; Gazova, Zuzana; von Bergen, Martin; Khlistunova, Inna; Wang, Yipeng; Hascher, Antje; Mandelkow, Eva-Maria; Biernat, Jacek; Mandelkow, Eckhard

2005-02-04

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.

Antiaging action of retinol: from molecular to clinical.

PubMed

Bellemère, G; Stamatas, G N; Bruère, V; Bertin, C; Issachar, N; Oddos, T

2009-01-01

The antiaging efficacy of retinol (ROL) has been explored mainly clinically in photoprotected skin sites and for high doses of ROL (0.4-1.6%). The objective of the study was to demonstrate the antiaging action of a low and tolerable dose of ROL (0.1%) ex vivo by measuring the expression of cellular retinoic-acid-binding protein II (CRABP2) and heparin-binding epidermal growth factor (HBEGF) by a histological evaluation of the epidermis and in vivo by assessing major aging signs and performing three-dimensional profilometry and digital imaging during a 9-month double-blind placebo-controlled study involving 48 volunteers. Finally, epidermal cell proliferation was evaluated using tryptophan fluorescence spectroscopy. Our results demonstrate that 0.1% ROL induced CRABP2 and HBEGF gene expression and increased keratinocyte proliferation and epidermal thickness. In human volunteers, topical application of a ROL-containing product improved all major aging signs assessed in our study (wrinkles under the eyes, fine lines and tone evenness). Moreover, tryptophan fluorescence increased in the active-agent-treated group and not in the placebo-treated group, indicating that cell proliferation was accelerated in vivo. These data demonstrate that a product containing a low dose (0.1%) of ROL promotes keratinocyte proliferation ex vivo and in vivo, induces epidermal thickening ex vivo and alleviates skin aging signs, without any significant adverse reaction. Copyright 2009 S. Karger AG, Basel.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

PubMed

Kumar, Sandeep; Jain, Kavish Kumar; Singh, Anupam; Panda, Amulya K; Kuhad, Ramesh Chander

2015-06-01

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry. Copyright © 2014 Elsevier Inc. All rights reserved.

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Fluorescence characterization of dissolved organic matter in the East China Sea after diatom red tide dispersion].

PubMed

Zhuo, Peng-ji; Zhao, Wei-hong

2009-05-01

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Exmax/Exmax = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm (Peak S) and 280/320 nm (Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A)and 330-350/420-480 nm (Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of them. Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

Effects of Pulsed Electric Field (PEF) Treatment on Enhancing Activity and Conformation of α-Amylase.

PubMed

Tian, Mei-ling; Fang, Ting; Du, Mu-ying; Zhang, Fu-sheng

2016-04-01

To explore an efficient, safe, and speedy application of pulsed electric field (PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activity, property and kinetic parameters of α-amylase were investigated. Conformational transitions were also studied with the aid of circular dichroism (CD) and fluorescence spectra. The maximum enzymatic activity of α-amylase was obtained under 15 kV/cm electric field intensity and 100 mL/min flow velocity PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14% compared with control. The activation effect could last for 18 h at 4 °C. PEF treatment could widen the range of optimum temperature for α-amylase, however, it barely exerted any effect on the optimum pH. On the other hand, α-amylase treated by PEF showed an increase of Vmax, t1/2 and ΔG, whereas a decrease of Km and k were observed. Furthermore, it can be observed from fluorescence and CD spectra that PEF treatment had increased the number of amino acid residues, especially that of tryptophan, on α-amylase surface with enhanced α-helices by 34.76% and decreased random coil by 12.04% on α-amylase when compared with that of untreated. These changes in structure had positive effect on enhancing α-amylase activity and property.

A biophysical investigation on the binding of proflavine with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM studies.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2016-12-01

Interaction of proflavine with hemoglobin (Hgb) was studied employing spectroscopy, calorimetry, and atomic force microscopy. The equilibrium constant was found to be of the order 10 4 M -1 . The quenching of Hgb fluorescence by proflavine was due to the complex formation. Calculation of the molecular distance (r) between the donor (β-Trp37 of Hgb) and acceptor (proflavine) suggested that energy can be efficiently transferred from the β-Trp37 residue at the α1β2 interface of the protein to the dye. Proflavine induced significant secondary structural changes in Hgb. Synchronous fluorescence studies showed that proflavine altered the microenvironment around the tryptophan residues to a greater extent than the tyrosine residues. Circular dichroism spectral studies showed that proflavine caused significant reduction in the α-helical content of Hgb. The esterase activity assay further complemented the circular dichroism data. The Soret band intensity of Hgb decreased upon complexation. Differential scanning calorimetry and circular dichroism melting results revealed that proflavine induced destabilization of Hgb. The binding was driven by both positive entropy and negative enthalpy. Atomic force microscopy studies revealed that the essential morphological features of hemoglobin were retained in the presence of proflavine. Overall, insights on the photophysical aspects and energetics of the binding of proflavine with Hgb are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser probes of the potential energy landscapes and conformational isomerization dynamics of flexible biomolecules

NASA Astrophysics Data System (ADS)

Dian, Brian; Clarkson, Jasper; Zwier, Timothy

2003-03-01

Using a combination of 2-color resonant two-photon ionization (R2PI), laser-induced fluorescence excitation (LIF), resonant ion-dip infrared spectroscopy (RIDIRS), fluorescence-dip infrared spectroscopy (FDIRS), and UV-UV hole-burning spectroscopy, the conformational preferences of a series of flexible biomolecules, including melatonin, N-acetyl-tryptophan methyl amide (NATMA), and their close analogs, have been determined in a molecular beam. These molecules are sufficiently complex to have hundreds of conformational minima, yet small enough that their potential energy landscapes can be explored in some detail. Once the conformational preferences of the molecules are established, these molecules are then studied using infrared-ultraviolet hole-filling and IR-induced population transfer spectroscopy. These methods utilize selective infrared excitation of single conformations of the molecule in the early portions of a gas-phase expansion, followed by collisional re-cooling of the excited population into its conformational minima for subsequent conformation-specific detection. Efficient isomerization is induced by the infrared excitation that redistributes population between the same conformations that have population in the absence of infrared excitation. Examples will be given in which the quantum yields for transfer of the population into the various conformational minima depend both on which conformation is excited and on which hydride stretch vibration is excited within a given conformation; that is, they are both conformation-selective and mode-selective.

Assessment of the unidentified organic matter fraction in fogwater using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Valsaraj, K.; Birdwell, J.

2010-07-01

Dissolved organic matter (DOM) in fogwaters from southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix (EEM) fluorescence spectroscopy. The results demonstrate that fluorescence spectroscopy can be used to obtain a qualitative assessment of the large fraction of fogwater organic carbon (~40 - 80% by weight) that cannot be identified in terms of specific chemical compounds. The method has the principle advantage that it can be applied at natural abundance concentrations, thus eliminating the need for large sample volumes required to isolate DOM for characterization by other spectroscopic (NMR, FTIR) and chemical (elemental) analyses. It was anticipated that the fogwater organic matter fluorescence spectra would resemble those of surface and rain waters, containing peaks indicative of both humic substances and fluorescent amino acids. Humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices had values comparable to other natural waters. Biological character (intensity of tyrosine and tryptophan peaks) was found to increase with organic carbon concentration. Fogwater organic matter appears to contain a mixture of terrestrially- and microbially-derived material. The fluorescence results show that most of the unidentified fogwater organic carbon can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems.

Effect of solvents on the fluorescence spectra of bacterial luciferase

NASA Astrophysics Data System (ADS)

Sukovataya, Irina E.; Tyulkova, Natalya A.; Kaykova, Elisaveta V.

2006-08-01

Bacteria luciferases catalyze the oxidation reaction of the long-chain aliphatic aldehyde and reduced flavinmononucleotide involving molecular oxygen to a respective fatty acid emitting light quanta in the visible spectrum. Fluorescence emission of luciferases from Photobacterium leiognathi dissolved in organic solvent-water mixtures was investigated. Methanol, acetone, dimethyl sulfoxide and formamide were used as organic solvents. As the methanol and acetone concentration is increased the emission maximum peak is decrease. In contrast, with dimethyl sulfoxide and formamide addition induced a increasing of the emission maximum intensity. The values of wavelength maximum (λ max) at the addition of this solvent can shows the spectra shifted to the red by about 12 nm. These increasing in the fluorescence intensity and in the λ max may be due to luciferase denaturation, resulting from the more intensive contact of chromospheres of luciferase with the solvent. At all used concentrations of methanol, acetone and formamide the shape of the fluorescence spectra was not changed. These studies demonstrate that the luciferase tryptophan fluorescence is sensitive to changes of physical-chemical property of enzyme environment. A comparison of activation/inactivation and fluorescence spectra of luciferase in methanol or acetone solutions shows that the extent of inactivation is larger than the extent of fluorescence changes at the same methanol or acetone concentration.

Accumulation and interaction of hypericin in low-density lipoprotein--a photophysical study.

PubMed

Mukherjee, Prasun; Adhikary, Ramkrishna; Halder, Mintu; Petrich, Jacob W; Miskovsky, Pavol

2008-01-01

The accumulation and interaction of hypericin with the biologically important macromolecule, low-density lipoprotein (LDL), is investigated using various steady-state and time-resolved fluorescence measurements. It is concluded that multiple hypericins can penetrate considerably deeply into the LDL molecule. Up to approximately 20 nonaggregated hypericin molecules can enter LDL; but upon increasing the hypericin concentration, the fluorescence lifetime of hypericin decreases drastically, suggesting most likely the self-quenching of aggregated hypericin. There is also evidence of energy transfer from tryptophans of the constituent protein, apoB-100, to hypericin in LDL. The results demonstrate the ability of LDL to solubilize hypericin (a known photosensitizer) in nonaggregated form, which has implications for the construction of drug delivery systems.

Excited state complex formation between methyl glyoxal and some aromatic bio-molecules: a fluorescence quenching study

NASA Astrophysics Data System (ADS)

Banerjee, D.; Mandal, A.; Mukherjee, S.

2003-01-01

Fluorescence quenching of some important aromatic bio-molecules (ABM) such as 3-aminophthalhydrazide (luminol), tryptophan (Try), phenylalanine and tyrosine (Tyr) by methyl glyoxal (MG) has been studied employing different spectroscopic techniques. The interaction of MG with ABM in the excited state has been analysed using Stern-Volmer (S-V) mechanism. In the case of MG-luminol system time correlated single photon counting (TCSPC) technique has also been applied to explain the S-V mechanism. The bimolecular rate constants obtained are found to be higher than the rate constant for diffusion controlled process. A plausible explanation of the quenching mechanism has been discussed on the basis of hydrogen bonding, charge transfer and energy transfer interaction between the colliding species.

Visible continuum pulses based on enhanced dispersive wave generation for endogenous fluorescence imaging.

PubMed

Cui, Quan; Chen, Zhongyun; Liu, Qian; Zhang, Zhihong; Luo, Qingming; Fu, Ling

2017-09-01

In this study, we demonstrate endogenous fluorescence imaging using visible continuum pulses based on 100-fs Ti:sapphire oscillator and a nonlinear photonic crystal fiber. Broadband (500-700 nm) and high-power (150 mW) continuum pulses are generated through enhanced dispersive wave generation by pumping femtosecond pulses at the anomalous dispersion region near zero-dispersion wavelength of high-nonlinear photonic crystal fibers. We also minimize the continuum pulse width by determining the proper fiber length. The visible-wavelength two-photon microscopy produces NADH and tryptophan images of mice tissues simultaneously. Our 500-700 nm continuum pulses support extending nonlinear microscopy to visible wavelength range that is inaccessible to 100-fs Ti:sapphire oscillators and other applications requiring visible laser pulses.

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

PubMed Central

Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

2014-01-01

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

2D fluorescence spectra measurement of six kinds of bioagents simulants by short range Lidar

NASA Astrophysics Data System (ADS)

Sanpedro, Man

2018-02-01

Pantoea agglomerans (Pan), Staphylococcus aureus (Sta), Bacillus globigii (BG) and Escherichia coli (EH), these four kinds of bioagents simulants of were cultured and then their growth curves were measured, the generation time was 0.99h, 0.835h, 1.07h and 1.909h, respectively. A small short range fluorescence lidar working at wavelengths of 266nm and 355nm was designed and used to measure the two-dimensional fluorescence spectra of bioagents simulants in the amino acid segment and NADH segment, respectively. In a controllable fluorescence measurement chamber, the two-dimensional fluorescence spectra of vegetative liquid bacterial aerosols as well as BSA and OVA, two protein toxinic simulants were measured with a resolution of 4nm. The two-dimensional fluorescence spectral shape of Pan, Sta, EH and BG, BSA and OVA were consistent with the standard fluorescent component tryptophan in the amino acid band with FWHM of 60nm, but the central wavelength of the fluorescence spectra of these simulants blue/purple shifted obviously as affected by the external biochemical environment, concentration and ratio of different bacterial internal fluorophores, so the energy level between the excited state and the ground state of the fluorescence molecule increased. Differently, weak NADH fluorescence spectra with 100nm FWHM inside the four vegetative bacteria aerosols were detected, but Rayleigh scattering, Raman scattering contribution of water, nitrogen in the fluorescence spectra could not be effectively extracted. The second - order derivative fluorescence spectra of four simulants showed that the high - order processing and recognition of the fluorescence spectra was feasible.

Analysis of rainwater dissolved organic carbon compounds using fluorescence spectrophotometry

NASA Astrophysics Data System (ADS)

Muller, Catherine L.; Baker, Andy; Hutchinson, Robert; Fairchild, Ian J.; Kidd, Chris

Global rainwater dissolved organic carbon (DOC) flux was recently estimated as 430 × 10 12 g C yr -1, yet little is known about the wide range of chemical compounds present, their sources, temporal patterns of variation, and the subsequent impact on climate and the environment. Precipitation events were sampled in Birmingham, UK between April 2005 and May 2007. Rainwater DOC compounds were analysed using fluorescence spectrophotometry. Three fluorophores were identified: HUmic-LIke Substances (HULIS), TYrosine-LIke Substances (TYLIS) and TRYptophan-LIke Substances (TRYLIS). Peak fluorescence intensities and locations for each substance were examined, and their variations with various meteorological parameters were investigated. The mean HULIS fluorescence intensity from all events was 209 a.u. (with sample fluorescence ranging from 37 a.u. to 995 a.u); mean fluorescence intensity was 469 a.u. (214-988 a.u) and 265 a.u. (50-876 a.u.) for TYLIS and TRYLIS, respectively. Results indicate that highest HULIS fluorescence intensities are experienced during convective events and events of continental origin, suggesting terrestrial/anthropogenic sources. Under well-mixed conditions, HULIS fluorescence intensity decreases, whereas during low wind speed, stagnation of the atmosphere results in higher fluorescence intensities, attributed to a build up of localised sources, particularly anthropogenic. TYLIS and TRYLIS did not show any significant trends for the meteorological variables. Fluorescence spectrophotometry is a fast, non-invasive technique which is demonstrated to be a powerful means of fingerprinting rainfall DOC compounds in real time for small sample volumes.

Efficient delivery of cell impermeable phosphopeptides by a cyclic peptide amphiphile containing tryptophan and arginine.

PubMed

Nasrolahi Shirazi, Amir; Tiwari, Rakesh Kumar; Oh, Donghoon; Banerjee, Antara; Yadav, Arpita; Parang, Keykavous

2013-05-06

Phosphopeptides are valuable reagent probes for studying protein-protein and protein-ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F'-GpYLPQTV, F'-NEpYTARQ, F'-AEEEIYGEFEAKKKK, F'-PEpYLGLD, F'-pYVNVQN-NH2, and F'-GpYEEI (F' = fluorescein), was evaluated in the presence or absence of a [WR]4, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]4 improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F'-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]4 and was found to be time-dependent. Confocal microscopy of a mixture of F'-PEpYLGLD and [WR]4 in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F'-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR]4. TEM results showed that the mixture of PEpYLGLD and [WR]4 formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]4 and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]4 can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides.

Porphyrin mediated photo-modification of the structure and function of human serum albumin

NASA Astrophysics Data System (ADS)

Rozinek, Sarah C.

Photosensitization reactions involve irradiating (with visible light) molecules with a high efficiency for either electron transfer or entering an excited triplet state (photosensitizer). Such reactions are applied to photodynamic cancer therapy, many medical laser-treatments, and a potential array of disinfection and pest elimination techniques. To understand the biophysical mechanisms of how these applications are effective at the protein level, the group of Dr. Brancaleon (UTSA) has investigated the irradiation of several dye-protein combinations, and discovered effects on protein structure and function. To further that work, we have investigated irradiation of the protein, human serum albumin (HSA), photosensitized by either protoporphyrin IX (PPIX) or meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP). HSA is the most abundant plasma protein, making it a likely substrate in PDT, and it possesses a specific binding pocket for iron-PPIX (heme) and possibly other porphyrin derivatives. The results of our research are summarized as follows. First, a thorough characterization of the binding of each photosensitizer to albumin was completed, elucidating a probable binding location for TSPP. Next, fluorescence lifetime emission of the single tryptophan residue, alongside circular dichroism, found tertiary structural changes around tryptophan and an overall 20% decrease in protein secondary structure after irradiation with TSPP bound. Finally, to determine if protein function was lost after photosensitization, size exclusion chromatography found modified albumin still recognizable by its receptor-protein, and comparative ex vivo up-take studies revealed that modified albumin is not processed the same way as native albumin in live tapeworm larva (Mesocestoides corti). Thus we found that visible light can induce partial unfolding of a protein by using a photo-activated ligand. These small structural modifications were sufficient to affect the protein's biological function.

Investigation of prostate cancer cells using NADH and Tryptophan as biomarker: multiphoton FLIM-FRET microscopy

NASA Astrophysics Data System (ADS)

Rehman, Shagufta; O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Chandra, Dhyan; Periasamy, Ammasi

2016-03-01

Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.

[Effects of a short-term diet of precooked corn flour on the vaginal cycle, in rats placed in various conditions of environmental illumination].

PubMed

Lopez de Onate, R; Giammanco, S; Carollo, F; Ernandes, M; Paderni, M A

1989-03-01

The aim of this research is to study the effects of a diet almost devoid of tryptophan, which is given by a feeding with precooked yellow corn meal (corn mush), on the alterations of the estrous cycle of animals in several conditions of environmental lighting. Indeed, it is known that cerebral serotonin influences the releasing of LH and consequently the ovulation. The different types of environmental lighting are: 1) Natural (alternating Day-Night = L/D). 2) Continuous dark (D/D). 3) Continuous light by sodium steams (L/L sodium). 4) Continuous light by fluorescent neon tubes (L/L neon). The muricide behaviour is studied by comparison rat-mouse. The feeding with precooked yellow corn meal (diet lacking of tryptophan) unchains in the 100% of the observations the CEA (Constant Estrous Anovulatory), and significantly shrinks the estral cycle in the female Wistar Rat in several conditions of environmental lighting.

Effect of the cooking method (grilling, roasting, frying and sous-vide) on the oxidation of thiols, tryptophan, alkaline amino acids and protein cross-linking in jerky chicken.

PubMed

Silva, Fábio A P; Ferreira, Valquíria C S; Madruga, Marta S; Estévez, Mario

2016-08-01

Broiler breast ( pectoralis major ) meat was submitted to salting with NaCl + NaNO 3 followed by a drying process to produce jerky-type chicken. The final product (raw broiler charqui) was desalted and then cooked using grilled, roasted, fried and sous-vide techniques. Sous-vide cooked samples showed lowest results of moisture loss compared to roasted and fried ones. Fatty acid profile suffered minor changes after cooking of broiler charqui. Regarding to protein oxidation, tryptophan fluorescence, protein carbonylation and disulphide bonds formation of chicken charqui were affected by cooking temperature while free thiol groups, Schiff base formation and hardness were mostly impacted by the length of cooking. Instrumental color of broiler charqui was affected by the type of cooking, being closely related with Maillard products formation. In conclusion, sous-vide technique seems to be the most advantageous cooking method to obtain high-quality ready-to-eat chicken charqui.

Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study

NASA Astrophysics Data System (ADS)

Singh, T. Sanjoy; Mitra, Sivaprasad

2011-03-01

Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern-Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant ( κq), Stern-Volmer constant ( KSV) and also the ligand-protein association constant ( Ka). The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 10 4 dm 3 mol -1. In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure.

Trehalose induced structural modulation of Bovine Serum Albumin at ambient temperature.

PubMed

Das, Ahana; Basak, Pijush; Pattanayak, Rudradip; Kar, Turban; Majumder, Rajib; Pal, Debadrita; Bhattacharya, Anindita; Bhattacharyya, Maitree; Banik, Samudra Prosad

2017-12-01

Trehalose is a well-known protein stabilizing osmolyte. The present study has been designed to understand the interaction of trehalose with BSA at ambient temperature. Steady state fluorescence and life-time analysis along with CD, DLS and ITC have been employed to show that trehalose causes surface-associated structural perturbation of BSA to promote its compaction. Trehalose at 0.1M concentration resulted in increased solvent exposure of one of the two tryptophans of BSA with a 5nm redshift in emission and enhanced susceptibility to acrylamide quenching with an increase in K SV from 2.61M -1 to 5.16M -1 . 0.5M trehalose resulted in reduced accessibility of tryptophan and destabilization of ANS binding (Forster radius increased from 24Å to 27.36Å for tryptophan-ANS FRET) indicating shielding of BSA in trehalose matrix. Simultaneously, there was compaction of BSA as shown by increased alpha-helicity from 45.85% to 48.81%, decreased thioflavin-T binding and reduction in hydrodynamic radius from 9.69nm to 6.59nm. Trehalose induced solution viscosity resulted in significant decrease in binding affinity of BSA towards curcumin and resveratrol. The results are in unison with the preferential exclusion and vitrification models to explain protein stabilization by trehalose and also points at the structure-function trade-off of proteins in presence of trehalose. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera.

PubMed

Abbassi, Shakeel; Patel, Krunal; Khan, Bashir; Bhosale, Siddharth; Gaikwad, Sushama

2016-02-01

Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036 ns) and τ2 (3.473±0.12 ns) indicating two populations of tryptophan. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

PubMed

Rocha, Danilo D; Espejo, Vinson R; Rainier, Jon D; La Clair, James J; Costa-Lotufo, Letícia V

2015-09-01

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence spectroscopy as a tool for determining microbial quality in potable water applications.

PubMed

Cumberland, Susan; Bridgeman, John; Baker, Andy; Sterling, Mark; Ward, David

2012-01-01

Building on previous work where fluorescence spectroscopy has been used to detect sewage in rivers, a portable LED spectrophotometer was used for the first time to establish bacterial numbers in a range of water samples. A mixed-method approach was used with standard bacteria enumeration techniques on diluted river water and sewage works final effluent using a number of diluents (Ringer's solution, tap water and potable spring water). Fluorescence from uncultured dilutions was detected at a 280 nm excitation/360 nm emission wavelength (corresponding to the region of tryptophan and indole fluorescence) and compared with bacteria numbers on the same cultured sample. Good correlations were obtained for total coliforms, E. coli and heterotrophic bacteria with the portable LED spectrophotometer (R2 = 0.78, 0.72 and 0.81 respectively). The results indicate that the portable spectrophotometer could be applied to establish the quality of drinking water in areas of poor sanitation that are subject to faecal contamination, where infrastructure failure has occurred in the supply of clean drinking water. This would be particularly useful where laboratory facilities are not at hand.

Fluorescence spectroscopy using excitation and emission matrix for quantification of tissue native fluorophores and cancer diagnosis

NASA Astrophysics Data System (ADS)

Wu, Binlin; Gayen, S. K.; Xu, M.

2014-03-01

Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.

Absorption and fluorescence properties of colored dissolved organic matter in the Ross Sea during austral summer

NASA Astrophysics Data System (ADS)

D'Sa, E. J.; Kim, H. C.; Ha, S. Y.

2016-12-01

Colored dissolved organic matter (CDOM) spectral absorption and excitation-emission matrix (EEMs) fluorescence with parallel factor analysis (PARAFAC) were examined in the Ross Sea during a survey conducted on board the R/V Araon in the austral summer of 14/15. CDOM absorption at 355 nm ranged from 0.06 to 1.14 m-1 while spectral slope S calculated between 275-295 nm wavelength ranged from 18.83 to 33.32 µm-1 with water masses playing an important role in its variability. Spectral slope S decreased with increasing CDOM absorption indicating the strong role of photo-oxidation on CDOM abundance during the summer. PARAFAC analysis of EEM data identified two humic-like (terrestrial and marine-like) and a protein-like (tryptophan-like) component. The two humic-like components were well correlated with little variability spatially and across the water column ( 0-100 m) likely indicating more refractory material. The protein-like fluorescent component was relatively quite variable supporting the autochthonous production of this fluorescent component in the highly productive Ross Sea waters.

A spectroscopic study on the interaction between gold nanoparticles and hemoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garabagiu, Sorina, E-mail: sgarabagiu@itim-cj.ro

2011-12-15

Highlights: Black-Right-Pointing-Pointer The interaction was studied using UV-vis and fluorescence spectroscopy. Black-Right-Pointing-Pointer Gold nanoparticles quench the fluorescence emission of hemoglobin solution. Black-Right-Pointing-Pointer The binding and thermodynamic constants were calculated. Black-Right-Pointing-Pointer Major impact: electrochemical applications of the complex onto a substrate. -- Abstract: The interaction between horse hemoglobin and gold nanoparticles was studied using optical spectroscopy. UV-vis and fluorescence spectra show that a spontaneous binding process occurred between hemoglobin and gold nanoparticles. The Soret band of hemoglobin in the presence of gold nanoparticles does not show significant changes, which proves that the protein retained its biological function. A shift to longermore » wavelengths appears in the plasmonic band of gold nanoparticles upon the attachment of hemoglobin molecules. Gold nanoparticles quench the fluorescence emission of tryptophan residues in the structure of hemoglobin. The Stern-Volmer quenching constant, the binding constant and the number of binding sites were also calculated. Thermodynamic parameters indicate that the binding was mainly due to hydrophobic interactions.« less

Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E. coli for efficient tryptophan bioproduction.

PubMed

Chen, Lin; Chen, Minliang; Ma, Chengwei; Zeng, An-Ping

2018-05-05

The L-tryptophan (Trp) biosynthesis pathway is highly regulated at multiple levels. The three types of regulations identified so far, namely repression, attenuation, and feedback inhibition have greatly impacted our understanding and engineering of cellular metabolism. In this study, feed-forward regulation is discovered as a novel regulation of this pathway and explored for engineering Escherichia coli for more efficient Trp biosynthesis. Specifically, indole glycerol phosphate synthase (IGPS) of the multifunctional enzyme TrpC from E. coli is found to be feed-forward inhibited by anthranilate noncompetitively. Surprisingly, IGPS of TrpC from both Saccharomyces cerevisiae and Aspergillus niger was found to be feed-forward activated, for which the glutamine aminotransferase domain is essential. The anthranilate binding site of IGPS from E. coli is identified and mutated, resulting in more tolerant variants for improved Trp biosynthesis. Furthermore, expressing the anthranilate-activated TrpC from A. niger in a previously engineered Trp producing E. coli strain S028 made the strain more robust in growth and more efficient in Trp production in bioreactor. It not only increased the Trp concentration from 19 to 29 g/L within 42 h, but also improved the maximum Trp yield from 0.15 to 0.18 g/g in simple fed-batch fermentations, setting a new level to rationally designed Trp producing strains. The findings are of fundamental interest for understanding and re-designing dynamics and control of metabolic pathways in general and provide a novel target and solution to engineering of E. coli for efficient Trp production particularly. Copyright © 2018. Published by Elsevier Inc.

Dissolved organic matter fluorescence at wavelength 275/342 nm as a key indicator for detection of point-source contamination in a large Chinese drinking water lake.

PubMed

Zhou, Yongqiang; Jeppesen, Erik; Zhang, Yunlin; Shi, Kun; Liu, Xiaohan; Zhu, Guangwei

2016-02-01

Surface drinking water sources have been threatened globally and there have been few attempts to detect point-source contamination in these waters using chromophoric dissolved organic matter (CDOM) fluorescence. To determine the optimal wavelength derived from CDOM fluorescence as an indicator of point-source contamination in drinking waters, a combination of field campaigns in Lake Qiandao and a laboratory wastewater addition experiment was used. Parallel factor (PARAFAC) analysis identified six components, including three humic-like, two tryptophan-like, and one tyrosine-like component. All metrics showed strong correlation with wastewater addition (r(2) > 0.90, p < 0.0001). Both the field campaigns and the laboratory contamination experiment revealed that CDOM fluorescence at 275/342 nm was the most responsive wavelength to the point-source contamination in the lake. Our results suggest that pollutants in Lake Qiandao had the highest concentrations in the river mouths of upstream inflow tributaries and the single wavelength at 275/342 nm may be adapted for online or in situ fluorescence measurements as an early warning of contamination events. This study demonstrates the potential utility of CDOM fluorescence to monitor water quality in surface drinking water sources. Copyright © 2015 Elsevier Ltd. All rights reserved.

Assembly of the alpha-toxin-hexamer of Staphylococcus aureus in the liposome membrane.

PubMed

Ikigai, H; Nakae, T

1987-02-15

It has been shown that the access of the alpha-toxin of Staphylococcus aureus to the target membrane and assembly of the hexamer can be monitored independently by respectively measuring the fluorescence energy transfer from the tryptophan residue(s) of the toxin to the dansylated phosphatidylethanolamine in the liposome membrane and the fluorescence increment of the toxin at 336 nm (Ikigai, H., and Nakae, T., (1987) J. Biol. Chem. 262, 2150-2155). Measurement of these parameters under various conditions showed the following results: when phosphatidylcholine (PC) liposomes composed of saturated fatty acids were mixed with the toxin, the fluorescence energy transfer occurred below, at, and above the transition temperature of the lipid, but the change of fluorescence at 336 nm was never detectable; when PC-liposomes containing unsaturated fatty acids were used, both the fluorescence energy transfer and the fluorescence increment of 336 nm were observed. These results suggested that the toxin-membrane interaction occurs in PC-membranes containing saturated and/or unsaturated fatty acids and that the oligomerization occurs only in the presence of PC containing unsaturated fatty acid(s). This conclusion was supported by the results of quantitative determination of the toxin-hexamer assembly and leakage of carboxyfluorescein from PC-liposomes under conditions similar to the above.

Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and L-tryptophan.

PubMed

Chen, Zhi; Zhang, Wei; Wang, Liping; Fan, Huajun; Wan, Qiang; Wu, Xuehao; Tang, Xunyou; Tang, James Z

2015-09-01

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened from amino acids, β-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid-liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. © 2015 Wiley Periodicals, Inc.

Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

PubMed

Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

2008-05-01

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK.

PubMed

Chen, Bo-En; Lin, Min-Guan; Lo, Huei-Fen; Wang, Tzu-Fan; Chi, Meng-Chun; Lin, Long-Liu

2013-01-01

Site-directed mutagenesis together with biochemical and biophysical techniques were used to probe effects of single-tryptophan-incorporated mutations on a bacterial molecular chaperone, Bacillus licheniformis DnaK (BlDnaK). Specifically, five phenylalanine residues (Phe(120), Phe(174), Phe(186), Phe(378) and Phe(396)) of BlDnaK were individually replaced by single tryptophans, thus creating site-specific probes for the fluorescence analysis of the protein. The steady-state ATPase activity for BlDnaK, F120W, F174W, F186W, F378W, and F396W was determined to be 76.01, 52.82, 25.32, 53.31, 58.84, and 47.53 nmol Pi/min/mg, respectively. Complementation test revealed that the single mutation at codons 120, 186, and 378 of the dnaK gene still allowed an Escherichia coli dnaK756-Ts strain to grow at a stringent temperature of 44°C. Simultaneous addition of co-chaperones and NR-peptide did not synergistically stimulate the ATPase activity of F174W and F396W, and these two proteins were unable to assist the refolding of GdnHCl-denatured luciferase. The heat-induced denaturation of all variants could be fitted adequately to a three-state model, in agreement with the observation for the wild-type protein. By CD spectral analysis, GdnHCl-induced unfolding transition for BlDnaK was 1.51 M corresponding to ΔG(N-U) of 1.69 kcal/mol; however, the transitions for mutant proteins were 1.07-1.55 M equivalent to ΔG(N-U) of 0.94-2.93 kcal/mol. The emission maximum of single-tryptophan-incorporated variants was in the range of 333.2-335.8 nm. Acrylamide quenching analysis showed that the mutant proteins had a dynamic quenching constant of 3.0-4.2 M(-1). Taken together, these results suggest that the molecular properties of BlDnaK have been significantly changed upon the individual replacement of selected phenylalanine residues by tryptophan. Copyright © 2012 Elsevier B.V. All rights reserved.

UV-induced photochemical heterogeneity of dissolved and attached organic matter associated with cyanobacterial blooms in a eutrophic freshwater lake.

PubMed

Xu, Huacheng; Jiang, Helong

2013-11-01

Cyanobacterial blooms represent a significant ecological and human health problem worldwide. In aquatic environments, cyanobacterial blooms are actually surrounded by dissolved organic matter (DOM) and attached organic matter (AOM) that bind with algal cells. In this study, DOM and AOM fractionated from blooming cyanobacteria in a eutrophic freshwater lake (Lake Taihu, China) were irradiated with a polychromatic UV lamp, and the photochemical heterogeneity was investigated using fluorescence excitation-emission matrix (EEM)-parallel factor (PARAFAC) analysis and synchronous fluorescence (SF)-two dimensional correlation spectroscopy (2DCOS). It was shown that a 6-day UV irradiation caused more pronounced mineralization for DOM than AOM (59.7% vs. 41.9%). The EEM-PARAFAC analysis identified one tyrosine-, one humic-, and two tryptophan-like components in both DOM and AOM, and high component photodegradation rates were observed for DOM versus AOM (k > 0.554 vs. <0.519). Moreover, SF-2DCOS found that the photodegradation of organic matters followed the sequence of tyrosine-like > humic-like > tryptophan-like substances. Humic-like substances promoted the indirect photochemical reactions, and were responsible for the higher photochemical rate for DOM. The lower photodegradation of AOM benefited the integrality of cells in cyanobacterial blooms against the negative impact of UV irradiation. Therefore, the photochemical behavior of organic matter was related to the adaptation of enhanced-duration cyanobacterial blooms in aquatic environments. Copyright © 2013 Elsevier Ltd. All rights reserved.

SERS+MEF of the anti-tumoral drug emodin adsorbed on silver nanoparticles

NASA Astrophysics Data System (ADS)

Sevilla, Paz; De Llanos, Raquel; Domingo, Concepción; Sánchez-Cortés, Santiago; García-Ramos, José V.

2010-02-01

Metal nanostructures are known to amplify the spontaneous emission of fluorescent molecules by resonant coupling to external electromagnetic fields. We have used spectroscopy to characterize the structural properties of emodin molecules, a natural anthraquinone dye, and bovine serum albumin, the most abundant protein in plasma, in the presence of silver nanoparticles. Aggregation of emodin at pH=10 and pH=6 gives rise to SERS and MEF effects in silver colloid. We have obtained MEF spectra at acidic pH=2.9 using two different silver nanostructures. We have also studied the change in the secondary structure of bovine serum albumin adsorbed on metal nanoparticles surface. Circular dichroism, fluorescence emission and fluorescence lifetime measurements indicate an increase in the alfa-helical content of the protein and a change in the environment of the tryptophan residues that bury in the interior of the biomolecule. This variation on the secondary structure could have further influence in the binding of the drug to form transport and regulatory complexes.

Interaction of toxic azo dyes with heme protein: biophysical insights into the binding aspect of the food additive amaranth with human hemoglobin.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2015-05-30

A biophysical study on the interaction of the food colorant amaranth with hemoglobin was undertaken. Spectrophotometric and spectrofluorimetric studies proposed for an intimate binding interaction between the dye and the protein. The dye quenched the fluorescence of the protein remarkably and the mechanism of quenching was found to be static in nature. Synchronous fluorescence studies suggested that the polarity around the tryptophan residues was altered in the presence of amaranth whereas the polarity around tyrosine residues remained largely unaltered. 3D fluorescence, FTIR and circular dichroism results suggested that the binding reaction caused conformational changes in hemoglobin. The negative far-UV CD bands exhibited a significantly large decrease in magnitude in the presence of amaranth. From calorimetry studies it was established that the binding was driven by a large positive entropic contribution and a small but favorable enthalpy change. Copyright © 2015 Elsevier B.V. All rights reserved.

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

PubMed

Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

2017-07-01

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

Regulation of Staphylococcal Penicillinase Synthesis

PubMed Central

Imsande, John; Zyskind, Judith W.; Mile, Imre

1972-01-01

5-Methyl tryptophan was found to be an efficient inducer of penicillinase synthesis in Staphylococcus aureus. Addition of actinomycin D or tryptophan to the culture medium shuts off the 5-methyl tryptophan-induced synthesis of penicillinase with an apparent half-life of approximately 1 to 2 min, respectively. Hence, in the induction of penicillinase synthesis, 5-methyl tryptophan seems to function as a structural analogue of penicillin rather than by becoming incorporated in proteins and thereby creating faulty penicillinase repressor or antirepressor. This conclusion is supported by similarities in the structures of the two compounds as revealed by solid atomic models. The fact that S. aureus exposed to 14C-penicillin in the absence of protein synthesis failed to synthesize penicillinase at an increased level when cell growth was resumed strongly suggests that a protein involved in the regulation of penicillinase synthesis must be synthesized in the presence of the penicillinase inducer. In turn, this observation suggests that the penicillinase inducer promotes penicillinase synthesis by directing the penicillinase regulatory protein (i.e., the penicillinase antirepressor) to acquire a different conformation when it is synthesized in the presence of the penicillinase inducer. A working model for the regulation of penicillinase synthesis based on these and other data has been constructed and is presented. Images PMID:4333374

Inhibiting the photosensitized oxidation of anthracene and tryptophan by means of natural antioxidants

NASA Astrophysics Data System (ADS)

Aksenova, N. A.; Vyzhlova, E. N.; Malinovskaya, V. V.; Parfenov, V. V.; Solov'eva, A. B.; Timashev, P. S.

2013-08-01

It is shown that model reactions of photosensitized oxidation of anthracene and tryptophan can be used for evaluation and comparison of antioxidant activity of various classes of compounds. Inhibition of the oxidation of substrates in the presence of the familiar antioxidants tocopherol (vitamin E), ascorbic acid (vitamin C), and mixtures of these vitamins with methionine, and in the presence of reputed antioxidants dihydroquercetin and taurine, are considered. It is concluded that all of the above compounds except for taurine have antioxidant properties; i.e., they reduce the rate constants of the photosensitized oxidation of anthracene and tryptophan. It is found that the inhibition of oxidation is associated with the interaction between antioxidants and singlet oxygen. Analysis of the kinetic dependences of the photosensitized oxidation of substrates in the presence of antioxidants reveals that a mixture of vitamins inhibits the process most efficiently, and inhibition occurs at the initial stages due to more active interaction between singlet oxygen and vitamin C

Kynurenine Pathway Metabolism is Involved in the Maintenance of the Intracellular NAD+ Concentration in Human Primary Astrocytes

PubMed Central

Grant, Ross; Nguyen, Susan; Guillemin, Gilles

2010-01-01

Efficient synthesis of NAD+ is critical to maintaining cell viability in all organs of the body. However, little is known of the pathway(s) by which cells of the central nervous system produce NAD+. The aim of this study was to investigate the relationship, between tryptophan degradation via the kynurenine pathway (KP) and de novo NAD+ synthesis in human astrocytes, a major cell type within the brain. In this study we observed that inhibition of single enzymes of the KP resulted in significant decreases in NAD+ levels in astroglial cells after a 24 hr period. We also observed that astrocytes cultured in media deficient in tryptophan, nicotinic acid and nicotinamide resulted in a 50% decrease in NAD+ levels after 24 hrs. This decrease in NAD+ was partially restored by supplementation of the culture media with either tryptophan or kynurenine, or nicotinic acid or with supply of the salvage pathway precursor nicotinamide. PMID:22084595

Spectroscopic characterization of dissolved organic matter in coking wastewater during bio-treatment: full-scale plant study.

PubMed

Xu, Ronghua; Ou, Huase; Yu, Xubiao; He, Runsheng; Lin, Chong; Wei, Chaohai

2015-01-01

This paper taking a full-scale coking wastewater (CWW) treatment plant as a case study aimed to characterize removal behaviors of dissolved organic matter (DOM) by UV spectra and fluorescence excitation-emission matrix-parallel factor analysis (PARAFAC), and investigate the correlations between spectroscopic indices and water quality parameters. Efficient removal rates of chemical oxygen demand (COD), dissolved organic carbon (DOC) and total nitrogen (TN) after the bio-treatment were 91.3%, 87.3% and 69.1%, respectively. UV270 was proven to be a stable UV absorption peak of CWW that could reflect the mixture of phenols, heterocyclics, polynuclear aromatic hydrocarbons and their derivatives. Molecular weight and aromaticity were increased, and also the content of polar functional groups was greatly reduced after bio-treatment. Three fluorescent components were identified by PARAFAC: C1 (tyrosine-like), C2 (tryptophan-like) and C3 (humic-like). The removal rate of protein-like was higher than that of humic-like and C1 was identified as biodegradable substance. Correlation analysis showed UV270 had an excellent correlation with COD (r=0.921, n=60, P<0.01) and DOC (r=0.959, n=60, P<0.01) and significant correlation (r=0.875, n=60, P<0.01) was also found between C2 and TN. Therefore, spectroscopic characterization could provide novel insights into removal behaviors of DOM and potential to monitor water quality real-time during CWW bio-treatment.

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

PubMed

Raman, Rajeev; Ptak, Christopher P; Hsieh, Ching-Lin; Oswald, Robert E; Chang, Yung-Fu; Sharma, Yogendra

2013-07-09

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

A Better Understanding of Protein Structure and Function by the Synthesis and Incorporation of Selenium- and Tellurium Containing Tryptophan Analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helmey, Sherif Samir; Rice, Ambrose Eugene; Hatch, Duane Michael

Unnatural heavy metal-containing amino acid analogs have shown to be very important in the analysis of protein structure, using methods such as X-ray crystallography, mass spectroscopy, and NMR spectroscopy. Synthesis and incorporation of selenium-containing methionine analogs has already been shown in the literature however with some drawbacks due to toxicity to host organisms. Thus synthesis of heavy metal tryptophan analogs should prove to be more effective since the amino acid tryptophan is naturally less abundant in many proteins. For example, bioincorporation of β-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]SeTrp) and β-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]SeTrp) has been shown in the following proteins without structural or catalytic perturbations: humanmore » annexin V, barstar, and dihydrofolate reductase. The reported synthesis of these Se-containing analogs is currently not efficient for commercial purposes. Thus a more efficient, concise, high-yield synthesis of selenotryptophan, as well as the corresponding, tellurotryptophan, will be necessary for wide spread use of these unnatural amino acid analogs. This research will highlight our progress towards a synthetic route of both [6,7]SeTrp and [6,7]TeTrp, which ultimately will be used to study the effect on the catalytic activity of Lignin Peroxidase (LiP).« less

Study of interactions of an anticancer drug neratinib with bovine serum albumin: Spectroscopic and molecular docking approach

NASA Astrophysics Data System (ADS)

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-03-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach.

PubMed

Wani, Tanveer A; Bakheit, Ahmed H; Abounassif, M A; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach

PubMed Central

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes. PMID:29564326

Human serum albumin stability and toxicity of anthraquinone dye alizarin complexone: an albumin-dye model.

PubMed

Ding, Fei; Zhang, Li; Diao, Jian-Xiong; Li, Xiu-Nan; Ma, Lin; Sun, Ying

2012-05-01

The complexation between the primary vector of ligands in blood plasma, human serum albumin (HSA) and a toxic anthraquinone dye alizarin complexone, was unmasked by means of circular dichroism (CD), molecular modeling, steady state and time-resolved fluorescence, and UV/vis absorption measurements. The structural investigation of the complexed HSA through far-UV CD, three-dimensional and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing with a reduction of α-helix upon conjugation. From molecular modeling and competitive ligand binding results, Sudlow's site I, which was the same as that of warfarin-azapropazone site, was appointed to retain high-affinity for alizarin complexone. Moreover, steady state fluorescence displayed that static type and Förster energy transfer is the operational mechanism for the vanish in the tryptophan (Trp)-214 fluorescence, this corroborates time-resolved fluorescence that HSA-alizarin complexone adduct formation has an affinity of 10(5) M(-1), and the driving forces were found to be chiefly π-π, hydrophobic, and hydrogen bonds, associated with an exothermic free energy change. These data should be utilized to illustrate the mechanism by which the toxicological action of anthraquinone dyes is mitigated by transporter HSA. Copyright © 2012 Elsevier Inc. All rights reserved.

Study on the interaction of a cyanine dye with human serum transferrin.

PubMed

Zhang, Xiu-feng; Chen, Lei; Yang, Qian-fan; Li, Qian; Sun, Xiao-ran; Chen, Hong-bo; Yang, Guang; Tang, Ya-lin

2015-12-01

Complexation between the primary carrier of ligands in blood plasma, human serum transferrin (Tf), and a cyanine dye, 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-phenyl-thiacarbocyanine-triethylam monium salt (PTC) was investigated using fluorescence spectra, UV/Vis absorption spectra, synchronous fluorescence spectra, circular dichroism (CD) and molecular dynamic docking. The experimental results demonstrate that the formation of PTC-Tf complex is stabilized by van der Waal's interactions and hydrogen bonds, and the binding constants were found to be 8.55 × 10(6), 8.19 × 10(6) and 1.75 × 10(4) M(-1). Moreover, fluorescence experiments prove that the operational mechanism for the fluorescence quenching is static quenching and non-radiative energy transfer. Structural investigation of the PTC-Tf complexes via synchronous fluorescence spectra and CD showed that the structure of Tf became more stable with a major increase in the α-helix content and increased polarity around the tryptophan residues after PTC binding. In addition, molecular modeling highlights the residues located in the N-lobe, which retain high affinity for PTC. The mode of action of the PTC-Tf complex is illustrated by these results, and may provide an effective pathway for the transport and targeted delivery of antitumor agents. Copyright © 2015 John Wiley & Sons, Ltd.

Aggregation induced emission enhancement (AIEE) characteristics of quinoline based compound - A versatile fluorescent probe for pH, Fe(III) ion, BSA binding and optical cell imaging

NASA Astrophysics Data System (ADS)

Manikandan, Irulappan; Chang, Chien-Huei; Chen, Chia-Ling; Sathish, Veerasamy; Li, Wen-Shan; Malathi, Mahalingam

2017-07-01

Novel benzimidazoquinoline derivative (AVT) was synthesized through a substitution reaction and characterized by various spectral techniques. Analyzing the optical properties of AVT under absorption and emission spectral studies in different environments exclusively with respect to solvents and pH, intriguing characteristics viz. aggregation induced emission enhancement (AIEE) in the THF solvent and 'On-Off' pH sensing were found at neutral pH. Sensing nature of AVT with diverse metal ions and bovine serum albumin (BSA) was also studied. Among the metal ions, Fe3 + ion alone tunes the fluorescence intensity of AVT probe in aqueous medium from ;turn-on; to ;turn-off; through ligand (probe) to metal charge transfer (LMCT) mechanism. The probe AVT in aqueous medium interacts strongly with BSA due to Fluorescence Resonance Energy Transfer (FRET) and the conformational change in BSA was further analyzed using synchronous fluorescence techniques. Docking study of AVT with BSA reveals that the active site of binding is tryptophan residue which is also supported by the experimental results. Interestingly, fluorescent AVT probe in cells was examined through cellular imaging studies using BT-549 and MDA-MB-231 cells. Thus, the single molecule probe based detection of multiple species and stimuli were described.

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

PubMed

Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

2015-02-09

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Extended wavelength anisotropy resolved multidimensional emission spectroscopy (ARMES) measurements: better filters, validation standards, and Rayleigh scatter removal methods

NASA Astrophysics Data System (ADS)

Casamayou-Boucau, Yannick; Ryder, Alan G.

2017-09-01

Anisotropy resolved multidimensional emission spectroscopy (ARMES) provides valuable insights into multi-fluorophore proteins (Groza et al 2015 Anal. Chim. Acta 886 133-42). Fluorescence anisotropy adds to the multidimensional fluorescence dataset information about the physical size of the fluorophores and/or the rigidity of the surrounding micro-environment. The first ARMES studies used standard thin film polarizers (TFP) that had negligible transmission between 250 and 290 nm, preventing accurate measurement of intrinsic protein fluorescence from tyrosine and tryptophan. Replacing TFP with pairs of broadband wire grid polarizers enabled standard fluorescence spectrometers to accurately measure anisotropies between 250 and 300 nm, which was validated with solutions of perylene in the UV and Erythrosin B and Phloxine B in the visible. In all cases, anisotropies were accurate to better than ±1% when compared to literature measurements made with Glan Thompson or TFP polarizers. Better dual wire grid polarizer UV transmittance and the use of excitation-emission matrix measurements for ARMES required complete Rayleigh scatter elimination. This was achieved by chemometric modelling rather than classical interpolation, which enabled the acquisition of pure anisotropy patterns over wider spectral ranges. In combination, these three improvements permit the accurate implementation of ARMES for studying intrinsic protein fluorescence.

Characterization of dissolved organic matter in an urbanized estuary located in Northeastern Brazil.

PubMed

Arguelho, Maria de Lara Palmeira de Macedo; Alves, José do Patrocínio Hora; Monteiro, Adnívia Santos Costa; Garcia, Carlos Alexandre Borges

2017-06-01

The Sal River estuary, which is located in the state of Sergipe, Northeastern Brazil, stands out as an urban estuary, anthropogenically impacted by untreated and treated wastewater discharge. Synchronous fluorescence spectroscopy and measurement of dissolved organic carbon (DOC) were used for characterization of dissolved organic matter (DOM) in the estuarine water. Dissolved organic carbon concentrations ranged from 7.5 to 19.0 mg L -1 and, in general, the highest values were recorded during dry season. For both seasons (dry and rainy), DOC presented an inverse linear relationship with salinity, which indicates a conservative dilution of organic matter coming into the estuary. During rainy season, anthropogenic organic constituents and humic substances from land-based sources predominated in DOM composition, carried by river flow. Whereas during the dry season, it has been observed a significant increase of products generated by microbial degradation of anthropogenic organic matter. The relationships between fluorescence intensity and salinity suggest a conservative behavior during rainy season and a non-conservative behavior during dry season, with addition of fluorescent organic matter into the intermediate zone of the estuary. Photodegradation by action of sunlight caused a decrease in fluorescence intensity of humic and tryptophan-like constituents and the release of photoproducts, resulting in an increase in fluorescence intensity of protein-like constituents.

Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans

PubMed Central

Coburn, Cassandra; Allman, Erik; Mahanti, Parag; Benedetto, Alexandre; Cabreiro, Filipe; Pincus, Zachary; Matthijssens, Filip; Araiz, Caroline; Mandel, Abraham; Vlachos, Manolis; Edwards, Sally-Anne; Fischer, Grahame; Davidson, Alexander; Pryor, Rosina E.; Stevens, Ailsa; Slack, Frank J.; Tavernarakis, Nektarios; Braeckman, Bart P.; Schroeder, Frank C.; Nehrke, Keith; Gems, David

2013-01-01

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death. PMID:23935448

Spectroscopic investigation on assisted sonocatalytic damage of bovine serum albumin (BSA) by metronidazole (MTZ) under ultrasonic irradiation combined with nano-sized ZnO

NASA Astrophysics Data System (ADS)

Gao, Jingqun; Liu, Bin; Wang, Jun; Jin, Xudong; Jiang, Renzheng; Liu, Lijun; Wang, Baoxin; Xu, Yongnan

2010-11-01

The previous work proved that the bovine serum albumin (BSA) could be damaged under the combined action of ultrasonic irradiation and ZnO. In this work, the assisted sonocatalytic damage of BSA using metronidazole (MTZ) as a sensitizer was further investigated by means of UV-vis and fluorescence spectra. The results indicated that the adding of MTZ could obviously promote the sonocatalytic damage of BSA under ultrasonic irradiation in the presence of nano-sized ZnO powder. Furthermore, it was found that the damage degree of BSA was aggravated by some influencing factors except ionic kind and strength. In addition, the damage site of BSA was also studied with synchronous fluorescence technology. It was found that the damage site was mainly at tryptophan (Trp) residue.

Effects of Pressure on Stability of Biomolecules in Solutions Studied by Neutron Scattering

NASA Astrophysics Data System (ADS)

Bellissent-Funel, Marie-Claire-; Appavou, Marie-Sousai; Gibrat, Gabriel

Studies of the pressure dependence on protein structure and dynamics contribute not only to the basic knowledge of biological molecules but have also a considerable relevance in full technology, like in food sterilization and pharmacy. Conformational changes induced by pressure as well as the effects on the protein stability have been mostly studied by optical techniques (optical absorption, fluorescence, phosphorescence), and by NMR. Most optical techniques used so far give information related to the local nature of the used probe (fluorescent or phosphorescent tryptophan). Small angle neutron scattering and quasi-elastic neutron scattering provide essential complementary information to the optical data, giving quantitative data on change of conformation of soluble globular proteins such as bovine pancreatic trypsin inhibitor (BPTI) and on the mobility of protons belonging to the protein surface residues.

Assessment of drinking water quality at the tap using fluorescence spectroscopy.

PubMed

Heibati, Masoumeh; Stedmon, Colin A; Stenroth, Karolina; Rauch, Sebastien; Toljander, Jonas; Säve-Söderbergh, Melle; Murphy, Kathleen R

2017-11-15

Treated drinking water may become contaminated while travelling in the distribution system on the way to consumers. Elevated dissolved organic matter (DOM) at the tap relative to the water leaving the treatment plant is a potential indicator of contamination, and can be measured sensitively, inexpensively and potentially on-line via fluorescence and absorbance spectroscopy. Detecting elevated DOM requires potential contamination events to be distinguished from natural fluctuations in the system, but how much natural variation to expect in a stable distribution system is unknown. In this study, relationships between DOM optical properties, microbial indicator organisms and trace elements were investigated for households connected to a biologically-stable drinking water distribution system. Across the network, humic-like fluorescence intensities showed limited variation (RSD = 3.5-4.4%), with half of measured variation explained by interactions with copper. After accounting for quenching by copper, fluorescence provided a very stable background signal (RSD < 2.2%) against which a ∼2% infiltration of soil water would be detectable. Smaller infiltrations would be detectable in the case of contamination by sewage with a strong tryptophan-like fluorescence signal. These findings indicate that DOM fluorescence is a sensitive indicator of water quality changes in drinking water networks, as long as potential interferents are taken into account. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Characterisation of landfill leachate by EEM-PARAFAC-SOM during physical-chemical treatment by coagulation-flocculation, activated carbon adsorption and ion exchange.

PubMed

Oloibiri, Violet; De Coninck, Sam; Chys, Michael; Demeestere, Kristof; Van Hulle, Stijn W H

2017-11-01

The combination of fluorescence excitation-emission matrices (EEM), parallel factor analysis (PARAFAC) and self-organizing maps (SOM) is shown to be a powerful tool in the follow up of dissolved organic matter (DOM) removal from landfill leachate by physical-chemical treatment consisting of coagulation, granular activated carbon (GAC) and ion exchange. Using PARAFAC, three DOM components were identified: C1 representing humic/fulvic-like compounds; C2 representing tryptophan-like compounds; and C3 representing humic-like compounds. Coagulation with ferric chloride (FeCl 3 ) at a dose of 7 g/L reduced the maximum fluorescence of C1, C2 and C3 by 52%, 17% and 15% respectively, while polyaluminium chloride (PACl) reduced C1 only by 7% at the same dose. DOM removal during GAC and ion exchange treatment of raw and coagulated leachate exhibited different profiles. At less than 2 bed volumes (BV) of treatment, the humic components C1 and C3 were rapidly removed, whereas at BV ≥ 2 the tryptophan-like component C2 was preferentially removed. Overall, leachate treated with coagulation +10.6 BV GAC +10.6 BV ion exchange showed the highest removal of C1 (39% - FeCl 3 , 8% - PACl), C2 (74% - FeCl 3 , 68% - PACl) and no C3 removal; whereas only 52% C2 and no C1 and C3 removal was observed in raw leachate treated with 10.6 BV GAC + 10.6 BV ion exchange only. Analysis of PARAFAC-derived components with SOM revealed that coagulation, GAC and ion exchange can treat leachate at least 50% longer than only GAC and ion exchange before the fluorescence composition of leachate remains unchanged. Copyright © 2017 Elsevier Ltd. All rights reserved.

Physical-biogeochemical Interactions in Northwestern Mediterranean, using the Glider "Sea Explorer" fitted with a Newly Developed Fluorescence Sensor, the MiniFluo-UV.

NASA Astrophysics Data System (ADS)

Goutx, M.; Besson, F.; Bhairy, N.; Germain, C.; Guigue, C.; Wassouf, G.; Beguery, L.; Tedetti, M.

2016-02-01

From July 2014 to February 2015, we deployed the SeaExplorer glider (ALSEAMAR Company, Meyreuil, France) fitted with the 2 optical pathways tryptophan-like (TRY-) and phenanthrenes-like (PHE-) MiniFluo-UV fluorescence sensor (french CNRS patent) and other sensors for hydrological and biogeochemical parameters (T, S, Chla, turbidity, humic-like) along the shelf off Marseille and the northern Mediterranean coast. Our goal was to characterize the distributions of dissolved organic matter (DOM) to assess its spatial variability in relation with biological and hydrological features, and human pressure. Interestingly, TRY-like and PHE-like concentrations displayed independent behavior along the bay of Marseille transects whereas they co-varied with physical and other biogeochemical descriptors near the mouth of the Rhone River. Around this highly anthropogenised area, lower salinities and higher concentrations in Chla, TRY- and PHE-like materials were observed. After rainy weather, waters of low salinity and of higher Chla, TRY- and PHE-like concentrations extended further southwards. Tryptophan-like concentrations showed a significant correlation with salinity and Chla indicating a biological source of DOM related to the Rhône inputs. Phenanthrene has different atypical profiles of other parameters showing patches of high concentrations at the surface throughout the transect (shipping) and at depth (sediment resuspension). The distribution of humic acid was controlled by the photo-bleaching. Southeast of the bay, intrusion of low temperature high salinity water in the surface layer was the likely feature of upwelled waters from subjacent layers. This study highlights the interest of coupling the outstanding maneuverability of SeaExplorer for exploring shallow waters and highly dynamic river mouth to the MiniFluo-UV sensor for analyzing the fluorescence properties of sea water. This work received fundings from the European Union's FP7 program within the framework of "NeXOS" project, for research, technological development and demonstration under grant agreement No 614102, in collaboration with ALSEAMAR Company.

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during L-tryptophan production in Escherichia coli.

PubMed

Liu, Lina; Chen, Sheng; Wu, Jing

2017-10-01

Escherichia coli FB-04(pta1), a recombinant L-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (L-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key L-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher L-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, L-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g -1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.

Fluorimetric Studies of a Trans-Membrane Protein and Its Interactions with Differently Functionalized Silver Nanoparticles.

PubMed

Gambucci, Marta; Tarpani, Luigi; Zampini, Giulia; Massaro, Giuseppina; Nocchetti, Morena; Sassi, Paola; Latterini, Loredana

2018-06-18

Trans-membrane proteins play important roles in the inter-cellular signaling to regulate the interactions among adjacent cells and influence cell fate. The study of the interactions between membrane proteins and nanomaterials is paramount for the design of nanomaterial-based therapies. In the present work, the fluorescence properties of the trans-membrane receptor Notch2 have been investigated. In particular, steady state and time resolved fluorescence methods have been used to characterize the emission of tryptophan residues of Notch2 and then this emission is used to monitor the impact of silver colloids on protein behavior. To this aim, silver colloids are prepared with two different methods to make sure they bear hydrophilic (citrate ions, C-AgNPs) or hydrophobic (dodecanethiol molecules D-AgNPs) capping agents; the preparation procedures are tightly controlled in order to obtain metal cores with similar size distributions (7.4 ± 2.5 and 5.0 ± 0.8 nm, respectively), thus making easier the comparison of the results. The occurrence of strong interactions between Notch2 and D-AgNPs is suggested by the efficient and statistically relevant quenching of the stationary protein emission already at low nanoparticle concentrations (ca. 12% quenching with [D-AgNPs] = 0.6nM). The quenching becomes even more pronounced (ca. 60%) when [D-AgNPs] is raised to 8.72nM. On the other hand, the addition of increasing concentrations of C-AgNPs to Notch2 does not affect the protein fluorescence (intensity variations below 5%) indicating that negligible interactions are taking place. The fluorescence data, recorded in the presence of increasing concentrations of silver nanoparticles, are then analyzed through the Stern-Volmer equation and the sphere of action model to discuss the nature of the interactions. The effect of D-AgNPs on the fluorescence decay times of Notch2 is also investigated and a decrease of the average decay time is observed (from 4.64 to 3.42 ns). The observed variations of the stationary and time-resolved fluorescence behavior of the protein are discussed in terms of static and collisional interactions. These results document that the capping shell is able to drive the protein-particle interactions, which have likely a hydrophobic nature.

Role of excited state solvent fluctuations on time-dependent fluorescence Stokes shift

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Tanping, E-mail: tanping@lsu.edu, E-mail: revatik@lsu.edu; Kumar, Revati, E-mail: tanping@lsu.edu, E-mail: revatik@lsu.edu

2015-11-07

We explore the connection between the solvation dynamics of a chromophore upon photon excitation and equilibrium fluctuations of the solvent. Using molecular dynamics simulations, fluorescence Stokes shift for the tryptophan in Staphylococcus nuclease was examined using both nonequilibrium calculations and linear response theory. When the perturbed and unperturbed surfaces exhibit different solvent equilibrium fluctuations, the linear response approach on the former surface shows agreement with the nonequilibrium process. This agreement is excellent when the perturbed surface exhibits Gaussian statistics and qualitative in the case of an isomerization induced non-Gaussian statistics. However, the linear response theory on the unperturbed surface breaksmore » down even in the presence of Gaussian fluctuations. Experiments also provide evidence of the connection between the excited state solvent fluctuations and the total fluorescence shift. These observations indicate that the equilibrium statistics on the excited state surface characterize the relaxation dynamics of the fluorescence Stokes shift. Our studies specifically analyze the Gaussian fluctuations of the solvent in the complex protein environment and further confirm the role of solvent fluctuations on the excited state surface. The results are consistent with previous investigations, found in the literature, of solutes dissolved in liquids.« less

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

Degradation of free tryptophan in a cookie model system and its application in commercial samples.

PubMed

Morales, Francisco J; Açar, Ozge C; Serpen, Arda; Arribas-Lorenzo, Gema; Gökmen, Vural

2007-08-08

The stability of free tryptophan (Trp) was examined in five cookie-resembling models at varying baking temperatures and durations. Trp was measured by HPLC coupled with a fluorescent detector. Trp degradation was significantly greater in cookies formulated with glucose compared with sucrose, regardless of the temperatures and durations of baking. A lag period was clearly observed in cookies formulated with sucrose. The type of sugar used in the dough formulation affected not only the thermal destruction kinetics but also the degree of degradation of free Trp. However, the type of leavening agent (ammonium bicarbonate versus sodium bicarbonate) did not affect the rate of Trp destruction as happens in Maillard-driven reactions. In addition, the free Trp content was analyzed in nine different flours and sixty-two commercial cookies, and it was found that free Trp varied from 0.4 to 1287.9 mg/kg for rice and wheat bran, respectively. It was found that free Trp was significantly higher in dietetic commercial samples formulated with wheat bran compared with other flours.

Spectral analysis of tissues from patients with cancer using a portable spectroscopic diagnostic ratiometer unit

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Pu, Yang; Sordillo, Peter P.; Budansky, Yury; Alfano, R. R.

2014-05-01

Spectral profiles of tissues from patients with breast carcinoma, malignant carcinoid and non-small cell lung carcinoma were acquired using native fluorescence spectroscopy. A novel spectroscopic ratiometer device (S3-LED) with selective excitation wavelengths at 280 nm and 335 nm was used to produce the emission spectra of the key biomolecules, tryptophan and NADH, in the tissue samples. In each of the samples, analysis of emission intensity peaks from biomolecules showed increased 340 nm/440 nm and 340 nm/460 nm ratios in the malignant samples compared to their paired normal samples. This most likely represented increased tryptophan to NADH ratios in the malignant tissue samples compared to their paired normal samples. Among the non-small cell lung carcinoma and breast carcinomas, it appeared that tumors of very large size or poor differentiation had an even greater increase in the 340 nm/440 nm and 340 nm/460 nm ratios. In the samples of malignant carcinoid, which is known to be a highly metabolically active tumor, a marked increase in these ratios was also seen.

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combining time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 100)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Trp was substituted either for leucine-31 ,located in the calcium binding loop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MIEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-31: a major local conformation corresponding to a lifetime class with a barycenter value of ~5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes ((tau)1 and (tau)2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the t4lifetime class at the expense of c2. A more restricted rotation of the Trp-31 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combinging time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 628)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Tip was substituted either for leucine-3 1 ,located in the calcium binding ioop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is. dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-3 1: a major local conformation corresponding to a lifetime class with a barycenter value of -5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes (τ1 and τ2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the τ4 lifetime class at the expense of c2. A more restricted rotation of the Trp-3 1 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Depressive disorder and gastrointestinal dysfunction after myocardial infarct are associated with abnormal tryptophan-5-hydroxytryptamine metabolism in rats

PubMed Central

Liu, Chunyan; Wang, Yangang

2017-01-01

In this study, we investigated the relationship between tryptophan-5-hydroxytryptamine metabolism, depressive disorder, and gastrointestinal dysfunction in rats after myocardial infarction. Our goal was to elucidate the physiopathologic bases of somatic/psychiatric depression symptoms after myocardial infarction. A myocardial infarction model was established by permanent occlusion of the left anterior descending coronary artery. Depression-like behavior was evaluated using the sucrose preference test, open field test, and forced swim test. Gastric retention and intestinal transit were detected using the carbon powder labeling method. Immunohistochemical staining was used to detect indoleamine 2,3-dioxygenase expression in the hippocampus and ileum. High-performance liquid chromatography with fluorescence and ultraviolet detection determined the levels of 5-hydroxytryptamine, its precursor tryptophan, and its metabolite 5-hydroxyindoleacetic acid in the hippocampus, distal ileum, and peripheral blood. All data were analyzed using one-way analyses of variance. Three weeks after arterial occlusion, rats in the model group began to exhibit depression-like symptoms. For example, the rate of sucrose consumption was reduced, the total and central distance traveled in the open field test were reduced, and immobility time was increased, while swimming, struggling and latency to immobility were decreased in the forced swim test. Moreover, the gastric retention rate and gastrointestinal transit rate were increased in the model group. Expression of indoleamine 2,3-dioxygenase was increased in the hippocampus and ileum, whereas 5-hydroxytryptamine metabolism was decreased, resulting in lower 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels in the hippocampus and higher levels in the ileum. Depressive disorder and gastrointestinal dysfunction after myocardial infarction involve abnormal tryptophan-5-hydroxytryptamine metabolism, which may explain the somatic, cognitive, and psychiatric symptoms of depression commonly observed after myocardial infarction. Peripheral 5-hydroxytryptamine is an important substance in the gut-brain axis, and its abnormal metabolism is a critical finding after myocardial infarct. PMID:28212441

Depressive disorder and gastrointestinal dysfunction after myocardial infarct are associated with abnormal tryptophan-5-hydroxytryptamine metabolism in rats.

PubMed

Lu, Xiaofang; Wang, Yuefen; Liu, Chunyan; Wang, Yangang

2017-01-01

In this study, we investigated the relationship between tryptophan-5-hydroxytryptamine metabolism, depressive disorder, and gastrointestinal dysfunction in rats after myocardial infarction. Our goal was to elucidate the physiopathologic bases of somatic/psychiatric depression symptoms after myocardial infarction. A myocardial infarction model was established by permanent occlusion of the left anterior descending coronary artery. Depression-like behavior was evaluated using the sucrose preference test, open field test, and forced swim test. Gastric retention and intestinal transit were detected using the carbon powder labeling method. Immunohistochemical staining was used to detect indoleamine 2,3-dioxygenase expression in the hippocampus and ileum. High-performance liquid chromatography with fluorescence and ultraviolet detection determined the levels of 5-hydroxytryptamine, its precursor tryptophan, and its metabolite 5-hydroxyindoleacetic acid in the hippocampus, distal ileum, and peripheral blood. All data were analyzed using one-way analyses of variance. Three weeks after arterial occlusion, rats in the model group began to exhibit depression-like symptoms. For example, the rate of sucrose consumption was reduced, the total and central distance traveled in the open field test were reduced, and immobility time was increased, while swimming, struggling and latency to immobility were decreased in the forced swim test. Moreover, the gastric retention rate and gastrointestinal transit rate were increased in the model group. Expression of indoleamine 2,3-dioxygenase was increased in the hippocampus and ileum, whereas 5-hydroxytryptamine metabolism was decreased, resulting in lower 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels in the hippocampus and higher levels in the ileum. Depressive disorder and gastrointestinal dysfunction after myocardial infarction involve abnormal tryptophan-5-hydroxytryptamine metabolism, which may explain the somatic, cognitive, and psychiatric symptoms of depression commonly observed after myocardial infarction. Peripheral 5-hydroxytryptamine is an important substance in the gut-brain axis, and its abnormal metabolism is a critical finding after myocardial infarct.

Development of Fluorescence Surrogates to Predict the Photochemical Transformation of Pharmaceuticals in Wastewater Effluents.

PubMed

Yan, Shuwen; Yao, Bo; Lian, Lushi; Lu, Xinchen; Snyder, Shane A; Li, Rui; Song, Weihua

2017-03-07

The photochemical transformation of pharmaceutical and personal care products (PPCPs) in wastewater effluents is an emerging concern for environmental scientists. In the current study, the photodegradation of 29 PPCPs was examined in effluents under simulated solar irradiation. Direct photodegradation, triplet state effluent organic matter ( 3 EfOM*)-mediated and hydroxyl radical (HO • )-mediated degradation are three major pathways in the removal process. With the photodegradation of trace levels of PPCPs, the excitation-emission matrix (EEM) fluorescence intensities of the effluents were also gradually reduced. Therefore, fluorescence peaks have been identified, for the first time, as appropriate surrogates to assess the photodegradation of PPCPs. The humic-like fluorescence peak is linked to direct photolysis-labile PPCPs, such as naproxen, ronidazole, diclofenac, ornidazole, tinidazole, chloramphenicol, flumequine, ciprofloxacin, methadone, and dimetridazole. The tyrosine-like EEM peak is associated with HO • /CO 3 •- -labile PPCPs, such as trimethoprim, ibuprofen, gemfibrozil, atenolol, carbamazepine, and cephalexin. The tryptophan-like peak is associated with 3 EfOM*-labile PPCPs, such as clenbuterol, metoprolol, venlafaxine, bisphenol A, propranolol, ractopamine, salbutamol, roxithromycin, clarithromycin, azithromycin, famotidine, terbutaline, and erythromycin. The reduction in EEM fluorescence correlates well with the removal of PPCPs, allowing a model to be constructed. The solar-driven removal of EEM fluorescence was applied to predict the attenuation of 11 PPCPs in five field samples. A close correlation between the predicted results and the experimental results suggests that fluorescence may be a suitable surrogate for monitoring the solar-driven photodegradation of PPCPs in effluents.

Threshold ionization spectroscopic investigation of supersonic jet-cooled, laser-desorbed Tryptophan

NASA Astrophysics Data System (ADS)

Taherkhani, Mehran; Armentano, Antonio; Černý, Jiří; Müller-Dethlefs, Klaus

2016-07-01

Tryptophan (Trp) was studied by two-colour Photoionization Efficiency (PIE) and Mass Analysed Threshold Ionization (MATI) spectroscopy using a laser desorption apparatus. Conformer A of Trp was excited into the S1 state (34,878 cm-1) and the second laser was scanned around the D0 cation ground and the D1 excited state. No ionization signal into the D0 state could be found, but a clear threshold was observed for the D1 state with an ionization energy of 66,704 ± 3 cm-1 (8.27 eV). This observation is explained in terms of the electronic configurations of the S1 and cationic states.

Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity.

PubMed

Zhai, Xiuhong; Malakhova, Margarita L; Pike, Helen M; Benson, Linda M; Bergen, H Robert; Sugár, István P; Malinina, Lucy; Patel, Dinshaw J; Brown, Rhoderick E

2009-05-15

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence

PubMed Central

Zhai, Xiuhong; Malakhova, Margarita L.; Pike, Helen M.; Benson, Linda M.; Bergen, H. Robert; Sugár, István P.; Malinina, Lucy; Patel, Dinshaw J.; Brown, Rhoderick E.

2009-01-01

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced “signature response,” i.e. ∼40% decrease in Trp intensity and ∼12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for ∼70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity. PMID:19270338

Maillard neoglycans as inhibitors for in vitro adhesion of F4+ enterotoxigenic Escherichia coli to piglet intestinal cells.

PubMed

Sarabia-Sainz, Héctor Manuel; Mata Haro, Verónica; Sarabia Sainz, José Andre-I; Vázquez-Moreno, Luz; Montfort, Gabriela Ramos-Clamont

2017-01-01

Adhesion of enterotoxigenic (ETEC) E. coli to host intestinal cells is mediated by lectin-like fimbriae that bind to specific glycan moieties on the surfaces of enterocytes. To prevent in vitro binding of E. coli F4 fimbriae (F4 ETEC + ) to piglet enterocytes, neoglycans were synthesized by the Maillard reaction conjugating lactose (Lac), galacto-oligosaccharides (GOS) or chitin oligosaccharides (Ochit) to porcine serum albumin (PSA). Neoglycans were characterized by SDS-PAGE, intrinsic tryptophan fluorescence and recognition by plant lectins, as well as by F4 ETEC variants. Electrophoretic patterns suggested the binding to PSA of 63, 13 and 2 molecules of Lac, GOS and Ochit, respectively. All neoglycans displayed quenching of tryptophan fluorescence consistent with the degree of glycation estimated by SDS-PAGE. Plant lectins recognized the neoglycans according to their specificity, whereas antigenic variants of F4 ETEC (ab, ac and ad) recognized PSA-Ochit and PSA-Lac with higher affinity than that for GOS. Neoglycans partially hindered the in vitro binding of F4 + ETEC to piglet enterocytes in a dose-dependent manner. The most effective blocking was observed with PSA-Lac that partially inhibited the adhesion of bacteria to enterocytes in a dose dependent manner, as quantified by flow cytometry. Increased production of the cytokines IL-6 and TNF-α was observed in response to F4 + ETEC infection of enterocytes and production was reduced in the presence of PSA-Ochit and PSA-GOS. These results suggest that neoglycans synthesized by the Maillard reaction could be useful in the prophylaxis of diarrhea in piglets.

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

Design and implementation of a dual-wavelength intrinsic fluorescence camera system

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Musacchia, Joseph J.; Gutierrez-Herrera, Enoch; Wang, Ying; Franco, Walfre

2017-03-01

Intrinsic UV fluorescence imaging is a technique that permits the observation of spatial differences in emitted fluorescence. It relies on the fluorescence produced by the innate fluorophores in the sample, and thus can be used for marker-less in-vivo assessment of tissue. It has been studied as a tool for the study of the skin, specifically for the classification of lesions, the delimitation of lesion borders and the study of wound healing, among others. In its most basic setup, a sample is excited with a narrow-band UV light source and the resulting fluorescence is imaged with a UV sensitive camera filtered to the emission wavelength of interest. By carefully selecting the excitation/emission pair, we can observe changes in fluorescence associated with physiological processes. One of the main drawbacks of this simple setup is the inability to observe more than a single excitation/emission pair at the same time, as some phenomena are better studied when two or more different pairs are studied simultaneously. In this work, we describe the design and the hardware and software implementation of a dual wavelength portable UV fluorescence imaging system. Its main components are an UV camera, a dual wavelength UV LED illuminator (295 and 345 nm) and two different emission filters (345 and 390 nm) that can be swapped by a mechanical filter wheel. The system is operated using a laptop computer and custom software that performs basic pre-processing to improve the image. The system was designed to allow us to image fluorescent peaks of tryptophan and collagen cross links in order to study wound healing progression.

Complete fluorescent fingerprints of extremophilic and photosynthetic microbes

NASA Astrophysics Data System (ADS)

Dartnell, Lewis R.; Storrie-Lombardi, Michael C.; Ward, John M.

2010-10-01

The work reported here represents a study into the total fluorescence exhibited by a broad selection of model, extremophilic and photosynthetic bacterial strains, over a great range of excitation and emission wavelengths from ultraviolet (UV) through visible to near infrared. The aim is to identify distinctive fluorescent features that may serve as detectable biosignatures of remnant microbial life on the Martian surface. A lab-bench fluorescence spectrometer was used to generate an excitation-emission matrix (EEM) for the unpigmented Escherichia coli, radiation-resistant Deinococcus radiodurans, Antarctic Dry Valley isolates Brevundimonas sp. MV.7 and Rhodococcus sp. MV.10, and the cyanobacterium Synechocystis sp. PCC 6803. Detailed EEMs, representing the fluorescence signature of each organism, are presented, and the most significant features suitable for biosignature surveys are identified, including small-molecule cellular metabolites, light-harvesting photosynthetic pigments and extracellular UV-screening compounds. E. coli exhibits the most intense emission from tryptophan, presumably due to the absence of UV-screening pigments that would shield the organism from short-wavelength light-exciting intracellular fluorescence. The efficacy of commonly available laser diodes for exciting cellular fluorescence is treated, along with the most appropriate filter wavelengths for imaging systems. The best combination of available laser diodes and PanCam filters aboard the ExoMars probe is proposed. The possibility of detecting fluorescence excited by solar UV radiation in freshly exposed surface samples by imaging when both sunlit and shadowed, perhaps by the body of the rover itself, is discussed. We also study how these biological fluorophore molecules may be degraded, and thus the potential biosignatures erased, by the high flux of far-ultraviolet light on Mars.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

PubMed

Tugaeva, Kristina V; Faletrov, Yaroslav V; Allakhverdiev, Elvin S; Shkumatov, Vladimir M; Maksimov, Eugene G; Sluchanko, Nikolai N

2018-02-26

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.

PubMed

Zuo, Ran; Zhang, Yi; Huguet-Tapia, Jose C; Mehta, Mishal; Dedic, Evelina; Bruner, Steven D; Loria, Rosemary; Ding, Yousong

2016-05-01

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Senescence-Induced Serotonin Biosynthesis and Its Role in Delaying Senescence in Rice Leaves1[C][W][OA

PubMed Central

Kang, Kiyoon; Kim, Young-Soon; Park, Sangkyu; Back, Kyoungwhan

2009-01-01

Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence. PMID:19439571

Effect of aminophylline on tryptophan and other aromatic amino acids in plasma, brain and other tissues and on brain 5-hydroxytryptamine metabolism.

PubMed

Curzon, G; Fernando, J C

1976-12-01

1 Aminophylline and other methylxanthines increase brain tryptophan and hence 5-hydroxytryptamine turnover. The mechanism of this effect of aminophylline was investigated. 2 At lower doses (greater than 100 mg/kg i.p.) the brain tryptophan increase could be explained by the lipolytic action of the drug, i.e. increased plasma unesterified fatty acid freeing plasma tryptophan from protein binding so that it became available to the brain. 3 Plasma unesterified fatty acid did not increase when aminophylline (109 mg/kg i.p.) was given to nicotinamide-treated rats but as both plasma total and free tryptophan rose, a tryptophan increase in the brain still occurred. 4 The rise in brain tryptophan concentration following the injection of a higher dose of the drug (150 mg/kg i.p.) could no longer be explained by a rise of plasma free tryptophan as the ratio of brain tryptophan to plasma free tryptophan rose considerably. Plasma total tryptophan fell and the plasma insulin concentration rose. 5 The increase of brain tryptophan concentration after injection of 150 mg/kg aminophylline appeared specific for this amino acid as brain tyrosine and phenyllanine did not increase. However as their plasma concentrations fell the brain/plasma ratio for all three amino acids rose. 6 The higher dose of aminophylline increased the muscle concentration of tryptophan but that of tyrosine fell and that of phenylalanine remained unaltered. The liver concentrations were not affected. 7 The aminophylline-induced increase of the ratio of brain tryptophan of plasma free tryptophan no longer occurred when the drug was given to animals injected with the beta-adrenoreceptor blocking agent propranolol or the diabetogenic agent streptozotocin. 8 The changes in brain tryptophan upon aminophylline injection may be explained by (a) increased availability of plasma tryptophan to the brain due to increased lipolysis and (b) increased effectiveness of uptake of tryptophan by the brain due to increased insulin secretion.

Kynurenine Pathway Pathologies: do Nicotinamide and Other Pathway Co-Factors have a Therapeutic Role in Reduction of Symptom Severity, Including Chronic Fatigue Syndrome (CFS) and Fibromyalgia (FM)

PubMed Central

Blankfield, Adele

2013-01-01

The definition of dual tryptophan pathways has increased the understanding of the mind-body, body-mind dichotomy. The serotonergic pathway highlights the primary (endogenous) psychiatric disorders. The up-regulation of the kynurenine pathway by physical illnesses can cause neuropathic and immunological disorders1 associated with secondary neuropsychiatric symptoms. Tryptophan and nicotinamide deficiencies fall within the protein energy malnutrition (PEM) spectrum. They can arise if the kynurenine pathway is stressed by primary or secondary inflammatory conditions and the consequent imbalance of available catabolic/anabolic substrates may adversely influence convalescent phase efficiency. The replacement of depleted or reduced NAD+ levels and other cofactors can perhaps improve the clinical management of these disorders. Chronic fatigue syndrome (CFS) and fibromyalgia (FM) appear to meet the criteria of a tryptophan-kynurenine pathway disorder with potential neuroimmunological sequelae. Aspects of some of the putative precipitating factors have been previously outlined.2,3 An analysis of the areas of metabolic dysfunction will focus on future directions for research and management. PMID:23922501

Palladium-Catalyzed Asymmetric Allylic Alkylation of 3-Substituted 1 H-Indoles and Tryptophan Derivatives with Vinylcyclopropanes.

PubMed

Trost, Barry M; Bai, Wen-Ju; Hohn, Christoph; Bai, Yu; Cregg, James J

2018-05-30

Vinylcyclopropanes (VCPs) are known to generate 1,3-dipoles with a palladium catalyst that initially serve as nucleophiles to undergo [3 + 2] cycloadditions with electron-deficient olefins. In this report, we reverse this reactivity and drive the 1,3-dipoles to serve as electrophiles by employing 3-alkylated indoles as nucleophiles. This represents the first use of VCPs for the completely atom-economic functionalization of 3-substituted 1 H-indoles and tryptophan derivatives via a Pd-catalyzed asymmetric allylic alkylation (Pd-AAA). Excellent yields and high chemo-, regio-, and enantioselectivities have been realized, providing various indolenine and indoline products. The method is amenable to gram scale and works efficiently with tryptophan derivatives that contain a diketopiperazine or diketomorpholine ring, allowing us to synthesize mollenine A in a rapid and ligand-controlled fashion. The obtained indolenine products bear an imine, an internal olefin, and a malonate motif, giving multiple sites with diverse reactivities for product diversification. Complicated polycyclic skeletons can be conveniently constructed by leveraging this unique juxtaposition of functional groups.

Probing the stereoselective interaction of ofloxacin enantiomers with corresponding monoclonal antibodies by multiple spectrometry

NASA Astrophysics Data System (ADS)

Mu, Hongtao; Xu, Zhenlin; Liu, Yingju; Sun, Yuanming; Wang, Baoling; Sun, Xiulan; Wang, Zhanhui; Eremin, Sergei; Zherdev, Anatoly V.; Dzantiev, Boris B.; Lei, Hongtao

2018-04-01

Although stereoselective antibody has immense potential in chiral compounds detection and separation, the interaction traits between stereoselective antibody and the corresponding antigenic enantiomers are not yet fully exploited. In this study, the stereospecific interactions between ofloxacin isomers and corresponding monoclonal antibodies (McAb-WR1 and McAb-MS1) were investigated using time-resolved fluorescence, steady-state fluorescence, and circular dichroism (CD) spectroscopic methods. The chiral recognition discrepancies of antibodies with ofloxacin isomers were reflected through binding constant, number of binding sites, driving forces and conformational changes. The major interacting forces of McAb-WR1 and McAb-MS1 chiral interaction systems were hydrophobic force and van der Waals forces joined up with hydrogen bonds, respectively. Synchronous fluorescence spectra and CD spectra results showed that the disturbing of tyrosine and tryptophan micro-environments were so slightly that no obvious secondary structure changes were found during the chiral hapten binding. Clarification of stereospecific interaction of antibody will facilitate the application of immunoassay to analyze chiral contaminants in food and other areas.

Characterizing fluorescent dissolved organic matter in a membrane bioreactor via excitation-emission matrix combined with parallel factor analysis.

PubMed

Maqbool, Tahir; Quang, Viet Ly; Cho, Jinwoo; Hur, Jin

2016-06-01

In this study, we successfully tracked the dynamic changes in different constitutes of bound extracellular polymeric substances (bEPS), soluble microbial products (SMP), and permeate during the operation of bench scale membrane bioreactors (MBRs) via fluorescence excitation-emission matrix (EEM) combined with parallel factor analysis (PARAFAC). Three fluorescent groups were identified, including two protein-like (tryptophan-like C1 and tyrosine-like C2) and one microbial humic-like components (C3). In bEPS, protein-like components were consistently more dominant than C3 during the MBR operation, while their relative abundance in SMP depended on aeration intensities. C1 of bEPS exhibited a linear correlation (R(2)=0.738; p<0.01) with bEPS amounts in sludge, and C2 was closely related to the stability of sludge. The protein-like components were more greatly responsible for membrane fouling. Our study suggests that EEM-PARAFAC can be a promising monitoring tool to provide further insight into process evaluation and membrane fouling during MBR operation. Copyright © 2016 Elsevier Ltd. All rights reserved.

An analysis of dissolved organic matter from freshwater Karelian Lakes using reversed-phase high-performance liquid chromatography with online absorbance and fluorescence analysis

NASA Astrophysics Data System (ADS)

Khundzhua, D. A.; Patsaeva, S. V.; Trubetskoj, O. A.; Trubetskaya, O. E.

2017-01-01

The spectral and optical properties of the fractionated components of dissolved organic matter (DOM) of three freshwater lakes in Karelia were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) with online detection of fluorescence and absorption spectra. It is shown that the DOM fractions are qualitatively similar, but differ quantitatively in the ratio of components and consist of at least three types of fluorophores: (1) hydrophilic "humic-like" fluorophore(s) with the emission maximum in the region of 420 nm and an absorption band at 260-270 nm; (2) hydrophobic "humic-like" fluorophore(s) with the emission maximum at approximately 450 nm that has no characteristic absorption maxima in the region from 220 to 400 nm; and (3) a "protein-like" fluorophore with the emission maximum in the region of 340-350 nm, which is typical of proteins and peptides containing tryptophan.

Antimicrobial peptides interact with peptidoglycan

NASA Astrophysics Data System (ADS)

Neelay, Om P.; Peterson, Christian A.; Snavely, Mary E.; Brown, Taylor C.; TecleMariam, Ariam F.; Campbell, Jennifer A.; Blake, Allison M.; Schneider, Sydney C.; Cremeens, Matthew E.

2017-10-01

Traditional therapeutics are losing effectiveness as bacterial resistance increases, and antimicrobial peptides (AMPs) can serve as an alternative source for antimicrobial agents. Their mode of action is commonly hypothesized to involve pore formation in the lipid membrane, thereby leading to cell death. However, bacterial cell walls are much more complex than just the lipid membrane. A large portion of the wall is comprised of peptidoglycan, yet we did not find any report of AMP-peptidoglycan interactions. Consequently, this work evaluated AMP-peptidoglycan and AMP-phospholipid (multilamellar vesicles) interactions through tryptophan fluorescence. Given that peptidoglycan is insoluble and vesicles are large particles, we took advantage of the unique properties of Trp-fluorescence to use one technique for two very different systems. Interestingly, melittin and cecropin A interacted with peptidoglycan to a degree similar to vancomycin, a positive control. Whether these AMP-peptidoglycan interactions relate to a killing mode of action requires further study.

Submillisecond Dynamics of Mastoparan X Insertion into Lipid Membranes.

PubMed

Schuler, Erin E; Nagarajan, Sureshbabu; Dyer, R Brian

2016-09-01

The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 μs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 μs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.

Retardation of Bulk Water Dynamics by Disaccharide Osmolytes

NASA Astrophysics Data System (ADS)

Shukla, Nimesh; Chen, Lee; Pomarico, Enrico; Chergui, Majed; Othon, Christina

Bioprotective nature of disaccharides is hypothesized to derive from the modification of the hydrogen bonding network of water which protects biomolecules through lowered water activity at the protein interface. Using ultrafast fluorescence spectroscopy, we measured the relaxation of bulk water dynamics around the induced dipole moment of two fluorescent probes (Lucifer Yellow Ethylenediamine and Tryptophan). Our results indicate a reduction in bulk water reorganization rate of approximately 30%. We observe this retardation in the low concentration regime measured at 0.1 and 0.25 M, far below the onset of glassy dynamics. This water structuring should be significant in crowded biological systems, contributing to a global change in protein energy landscape, resulting in a significant enhancement of protein stability under environmental stress. We observed similar dynamic reduction for two disaccharide osmolytes, sucrose and trehalose, with trehalose being the more effective in reducing solvation dynamics.

Comparison of the effect of UV laser radiation and of a radiomimetic substance on chromatin

NASA Astrophysics Data System (ADS)

Radulescu, Irina; Radu, Liliana; Serbanescu, Ruxandra; Nelea, V. D.; Martin, C.; Mihailescu, Ion N.

1998-07-01

The damages of the complex of deoxyribonucleic acid (DNA) and proteins from chromatin, produced by the UV laser radiation and/or by treatment with a radiomimetic substance, bleomycin, were compared. The laser radiation and bleomycin effects on chromatin structure were determined by the static and dynamic fluorimetry of chromatin complexes with the DNA specific ligand-- proflavine and by the analysis of tryptophan chromatin intrinsic fluorescence. Time resolved spectroscopy is a sensitive technique which allows to determine the excited state lifetimes of chromatin--proflavine complexes. Also, the percentage contributions to the fluorescence of proflavine, bound and unbound to chromatin DNA, were evaluated. The damages produced by the UV laser radiation on chromatin are similar with those of radiomimetic substance action and consists in DNA and proteins destruction. The DNA damage degree has been determined. The obtained results may constitute some indications in the laser utilization in radiochimiotherapy.

Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence

NASA Astrophysics Data System (ADS)

Bayram, Serene S.; Green, Philippe; Blum, Amy Szuchmacher

2018-04-01

We propose the use of a cysteine mutant of TMV coat protein as a signal transducer for the selective sensing and quantification of the heavy metal ions, Cd2+, Pb2+, Zn2+ and Ni2+ based on intrinsic tryptophan quenching. TMV coat protein is inexpensive, can be mass-produced since it is expressed and extracted from E-coli. It also displays several different functional groups, enabling a wide repertoire of bioconjugation chemistries; thus it can be easily integrated into functional devices. In addition, TMV-ion interactions have been widely reported and utilized for metallization to generate organic-inorganic hybrid composite novel materials. Building on these previous observations, we herein determine, for the first time, the TMV-ion binding constants assuming the static fluorescence quenching model. We also show that by comparing TMV-ion interactions between native and denatured coat protein, we can distinguish between chemically similar heavy metal ions such as cadmium and zinc ions.

Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

PubMed

Vuillard, L; Rabilloud, T; Goldberg, M E

1998-08-15

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

PubMed Central

Basu, S; Mandal, C; Allen, A K

1988-01-01

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin. Images Fig. 3. PMID:3140796

Tryptophan depletion and formation of alpha-aminoadipic and gamma-glutamic semialdehydes in porcine burger patties with added phenolic-rich fruit extracts.

PubMed

Ganhão, Rui; Morcuende, David; Estévez, Mario

2010-03-24

The effect of added fruit extracts on the oxidation of muscle proteins in porcine burger patties subjected to cooking and chill storage was studied. Extracts from arbutus berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses (Rosa canina L., RC), and elm-leaf blackberries (Rubus ulmifolius Schott, RU) were prepared, characterized, added to burger patties (3% of total weight), and evaluated as inhibitors of protein oxidation. Negative (no added extract, C) and positive control (added quercetin, 230 mg/kg, Q) groups were also included in the design. Protein oxidation was assessed by means of tryptophan loss using fluorescence spectroscopy (FS) and formation of the specific protein carbonyls alpha-aminoadipic (AAS) and gamma-glutamic semialdehyde (GGS) using liquid chromatography and mass spectroscopy (LC-MS). Both advanced methodologies (FS and LC-MS) were found to be reliable and specific protein oxidation measurements that allow us to gain chemical insight into protein oxidation. The mechanisms likely involved in the oxidative reactions affecting proteins during cooking and storage of burger patties are profusely discussed. Phenolic-rich fruit extracts protected tryptophan residues against oxidation and inhibited the formation of both semialdehydes in burger patties during cooking and subsequent chill storage. In general, RC, RU, and AU were the most effective inhibitors of protein oxidation, with this effect being more intense than that of pure polyphenols like quercetin. These fruit extracts could be considered functional ingredients as their antioxidant actions contribute to the enhancement of the nutritional value of the meat products.

Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

PubMed Central

Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

2013-01-01

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

PubMed

Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

2015-12-01

The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses. © 2015 The Protein Society.

Applicability of Fluorescence and Absorbance Spectroscopy to Estimate Organic Pollution in Rivers

PubMed Central

Knapik, Heloise Garcia; Fernandes, Cristovão Vicente Scapulatempo; de Azevedo, Júlio Cesar Rodrigues; do Amaral Porto, Monica Ferreira

2014-01-01

Abstract This article explores the applicability of fluorescence and absorbance spectroscopy for estimating organic pollution in polluted rivers. The relationship between absorbance, fluorescence intensity, dissolved organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), and other water quality parameters were used to characterize and identify the origin and the spatial variability of the organic pollution in a highly polluted watershed. Analyses were performed for the Iguassu River, located in southern Brazil, with area about 2,700 km2 and ∼3 million inhabitants. Samples were collect at six monitoring sites covering 107 km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285 nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies. PMID:25469076

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy.

PubMed

Portugal, Carla A M; Truckenmüller, Roman; Stamatialis, Dimitrios; Crespo, João G

2014-11-10

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell-scaffold interactions. The changes induced upon adsorption of two model proteins with different geometries, trypsin (globular conformation) and fibrinogen (rod-shaped conformation) on poly-l-lactic acid (PLLA) scaffolds with different surface topographies, flat, fibrous and surfaces with aligned nanogrooves, were assessed by fluorescence spectroscopy monitoring, using tryptophan as structural probe. Hence, the maximum emission blue shift and the increase of fluorescence anisotropy observed after adsorption of globular and rod-like shaped proteins on surfaces with parallel nanogrooves were ascribed to more intense protein-surface interactions. Furthermore, the decrease of fluorescence anisotropy observed upon adsorption of proteins to scaffolds with fibrous morphology was more significant for rod-shaped proteins. This effect was associated to the ability of these proteins to adjust to curved surfaces. The additional unfolding of proteins induced upon adsorption on scaffolds with a fibrous morphology may be the reason for better cell attachment there, promoting an easier access of cell receptors to initially hidden protein regions (e.g. RGDS sequence), which are known to have a determinant role in cell attaching processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectroscopic investigations on the effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots on catalase

NASA Astrophysics Data System (ADS)

Sun, Haoyu; Yang, Bingjun; Cui, Erqian; Liu, Rutao

2014-11-01

Quantum dots (QDs) are recognized as some of the most promising semiconductor nanocrystals in biomedical applications. However, the potential toxicity of QDs has aroused wide public concern. Catalase (CAT) is a common enzyme in animal and plant tissues. For the potential application of QDs in vivo, it is important to investigate the interaction of QDs with CAT. In this work, the effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak at 612 nm (QDs-612) on CAT was investigated by fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet-visible (UV-vis) absorption and circular dichroism (CD) techniques. Binding of QDs-612 to CAT caused static quenching of the fluorescence, the change of the secondary structure of CAT and the alteration of the microenvironment of tryptophan residues. The association constants K were determined to be K288K = 7.98 × 105 L mol-1 and K298K = 7.21 × 105 L mol-1. The interaction between QDs-612 and CAT was spontaneous with 1:1 stoichiometry approximately. The CAT activity was also inhibited for the bound QDs-612. This work provides direct evidence about enzyme toxicity of QDs-612 to CAT in vitro and establishes a new strategy to investigate the interaction between enzyme and QDs at a molecular level, which is helpful for clarifying the bioactivities of QDs in vivo.

Study on interaction between curcumin and pepsin by spectroscopic and docking methods.

PubMed

Ying, Ming; Huang, Fengwen; Ye, Haidong; Xu, Hong; Shen, Liangliang; Huan, Tianwen; Huang, Shitong; Xie, Jiangfeng; Tian, Shengli; Hu, Zhangli; He, Zhendan; Lu, Jun; Zhou, Kai

2015-08-01

The interaction between curcumin and pepsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD), and molecular docking. Under physiological pH value in stomach, the fluorescence of pepsin can be quenched effectively by curcumin via a combined quenching process. Binding constant (Ka) and binding site number (n) of curcumin to pepsin were obtained. According to the theory of Förster's non-radiation energy transfer, the distance r between pepsin and curcumin was found to be 2.45 nm within the curcumin-pepsin complex, which implies that the energy transfer occurs between curcumin and pepsin, leading to the quenching of pepsin fluorescence. Fluorescence experiments also suggest that curcumin is located more closely to tryptophan residues than tyrosine residues. CD spectra together with UV-vis absorbance studies show that binding of curcumin to pepsin results in the extension of peptide strands of pepsin with loss of some β-sheet structures. Thermodynamic parameters calculated from the binding constants at different temperatures reveal that hydrophobic force plays a major role in stabilizing the curcumin-pepsin complex. In addition, docking results support the above experimental findings and suggest the possible hydrogen bonds of curcumin with Thr-77, Thr-218, and Glu-287 of pepsin, which help further stabilize the curcumin-pepsin complex. Copyright © 2015 Elsevier B.V. All rights reserved.

Peroxynitrite-induced structural perturbations in human IgG: A physicochemical study.

PubMed

Arfat, Mir Yasir; Arif, Zarina; Chaturvedi, Sumit Kumar; Moinuddin; Alam, Khursheed

2016-08-01

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluorescence probe of polypeptide conformational dynamics in gas phase and in solution

NASA Astrophysics Data System (ADS)

Iavarone, Anthony T.; Meinen, Jan; Schulze, Susanne; Parks, Joel H.

2006-07-01

Fluorescence measurements of polypeptides derivatized with the fluorescent dye BODIPY TMR have been used to probe the polypeptide conformational dynamics as a function of temperature and charge state. Measurements of (BODIPY TMR)-[Pro]n-Arg-Trp and (BODIPY TMR)-[Gly-Ser]m-Arg-Trp have been performed for charge states 1+ and 2+ of n = 4 and 10 and m = 2 and 5. The 2+ charge states of both of these polypeptides exhibit similar temperature dependences for equal chain lengths (n = 4, m = 2 and n = 10, m = 5) and suggest conformations dominated by Coulomb repulsion. In the absence of such Coulomb repulsion, the 1+ charge state conformations appear to be characterized by the flexibility of the polypeptide chain for which [Gly-Ser]m > [Pro]n. Comparisons of these gas phase polypeptide measurements with corresponding measurements in solution provide a direct measure of the effects of solvent on the conformational dynamics. The change in fluorescence as a function of temperature in the gas phase is two orders of magnitude greater than that in solution, a dramatic result we attribute to the restrictions on intramolecular dynamics imposed by diffusion-limited kinetics and the lack of shielding by solvent. Measurements were also made of unsolvated Pron peptides without the tryptophan (Trp) residue to isolate the interaction of the fluorescent dye with charges.

The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity.

PubMed

Kroczynska, Barbara; Evangelista, Christina M; Samant, Shalaka S; Elguindi, Ebrahim C; Blond, Sylvie Y

2004-03-19

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

TRYPTOPHAN SYNTHETASE LEVELS IN ESCHERICHIA COLI, SHIGELLA DYSENTERIAE, AND TRANSDUCTION HYBRIDS

PubMed Central

Eisenstein, Richard B.; Yanofsky, Charles

1962-01-01

Eisenstein, Richard B. (Western Reserve University, Cleveland, Ohio) and Charles Yanofsky. Tryptophan synthetase levels in Escherichia coli, Shigella dysenteriae, and transduction hybrids. J. Bacteriol. 83:193–204. 1962—Shigella dysenteriae and Escherichia coli, strains K-12 and B, were found to produce low levels of tryptophan synthetase, although some hybrids, formed by the introduction of the gene cluster concerned with tryptophan synthesis from S. dysenteriae into E. coli, produced high levels of this enzyme system. A revertant obtained from a tryptophan-requiring mutant also formed high levels of tryptophan synthetase. The gene or genes responsible for high enzyme production in these strains was shown to be linked to the cluster of genes concerned with tryptophan synthesis. The cause of high enzyme production was investigated. Various lines of evidence, including stimulation of growth by tryptophan precursors, sensitivity to inhibition by 5-methyltryptophan, absence of accumulation of tryptophan, and repression of enzyme formation by anthranilic acid and tryptophan, suggested that high enzyme production in the strains examined results from a partial block in the tryptophan pathway and not from resistance to repression by tryptophan. The conversion of shikimic acid-5-phosphate to anthranilic acid appears to be the partially blocked reaction in the strains studied. PMID:13889700

Understanding the role of Arg96 in structure and stability of green fluorescent protein

PubMed Central

Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Shavlovsky, Michail M.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, Konstantin K.

2010-01-01

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP. PMID:18470931

Fluorescence peak integration ratio IC:IT as a new potential indicator tracing the compositional changes in chromophoric dissolved organic matter.

PubMed

Zhou, Yongqiang; Shi, Kun; Zhang, Yunlin; Jeppesen, Erik; Liu, Xiaohan; Zhou, Qichao; Wu, Huawu; Tang, Xiangming; Zhu, Guangwei

2017-01-01

The present study demonstrates that the ratio of fluorescence integration of peak C to peak T (I C :I T ) can be used as an indicator tracing the compositional dynamics of chromophoric dissolved organic matter (CDOM). CDOM absorption and fluorescence spectroscopy and stable isotope δ 13 C were determined on a seasonal basis in seventeen Chinese inland waters as well as in a series of mixing and photodegradation experiments in the lab. A strong positive linear correlation was recorded between I C :I T and the ratio of terrestrial humic-like C1 to tryptophan-like C4 (C1:C4) derived by parallel factor analysis. The r 2 for the linear fitting between I C :I T and C1:C4 (r 2 =0.80) was notably higher than between C1:C4 and other indices tested, including the ratio of CDOM absorption at 250nm to 365nm, i.e. a(250):a(365) (r 2 =0.09), spectral slope (S 275-295 ) (r 2 =0.26), spectral slope ratio (S R ) (r 2 =0.31), the humification index (HIX) (r 2 =0.47), the recent autochthonous biological contribution index (BIX) (r 2 =0.27), and a fluorescence index (FI 370 ) (r 2 =0.07). I C :I T exhibited larger variability than the remaining six indices and a closer correlation with stable isotope δ 13 C than that observed for a(250):a(365), S 275-295 , S R , FI 370 , and BIX during field campaigns. Confirming our field observations, significant correlations were recorded between I C :I T and the remaining six indices, and I C :I T also demonstrated notably larger variability than the six other indices during our wastewater addition experiment. Compared with HIX, eutrophic water addition and photobleaching substantially decreased I C :I T but had no pronounced effect on a(250):a(365), S 275-297 , S R , FI 370 , and BIX, further suggesting that I C :I T is the most efficient indicator of the CDOM compositional dynamics. Copyright © 2016 Elsevier B.V. All rights reserved.

Primary and secondary structure dependence of peptide flexibility assessed by fluorescence-based measurement of end-to-end collision rates.

PubMed

Huang, Fang; Hudgins, Robert R; Nau, Werner M

2004-12-22

The intrachain fluorescence quenching of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is measured in short peptide fragments, namely the two strands and the turn of the N-terminal beta-hairpin of ubiquitin. The investigated peptides adopt a random-coil conformation in aqueous solution according to CD and NMR experiments. The combination of quenchers with different quenching efficiencies, namely tryptophan and tyrosine, allows the extrapolation of the rate constants for end-to-end collision rates as well as the dissociation of the end-to-end encounter complex. The measured activation energies for fluorescence quenching demonstrate that the end-to-end collision process in peptides is partially controlled by internal friction within the backbone, while measurements in solvents of different viscosities (H2O, D2O, and 7.0 M guanidinium chloride) suggest that solvent friction is an additional important factor in determining the collision rate. The extrapolated end-to-end collision rates, which are only slightly larger than the experimental rates for the DBO/Trp probe/quencher system, provide a measure of the conformational flexibility of the peptide backbone. The chain flexibility is found to be strongly dependent on the type of secondary structure that the peptides represent. The collision rates for peptides derived from the beta-strand motifs (ca. 1 x 10(7) s(-1)) are ca. 4 times slower than that derived from the beta-turn. The results provide further support for the hypothesis that chain flexibility is an important factor in the preorganization of protein fragments during protein folding. Mutations to the beta-turn peptide show that subtle sequence changes strongly affect the flexibility of peptides as well. The protonation and charge status of the peptides, however, are shown to have no significant effect on the flexibility of the investigated peptides. The meaning and definition of end-to-end collision rates in the context of protein folding are critically discussed.

Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2016-11-15

Interaction of the food colorant tartrazine with human hemoglobin was studied using multispectroscopic and microcalorimetric techniques to gain insights into the binding mechanism and thereby the toxicity aspects. Hemoglobin spectrum showed hypochromic changes in the presence of tartrazine. Quenching of the fluorescence of hemoglobin occurred and the quenching mechanism was through a static mode as revealed from temperature dependent and time-resolved fluorescence studies. According to the FRET theory the distance between β-Trp37 of hemoglobin and bound tartrazine was evaluated to be 3.44nm. Synchronous fluorescence studies showed that tartrazine binding led to alteration of the microenvironment around the tryptophans more in comparison to tyrosines. 3D fluorescence and FTIR data provided evidence for conformational changes in the protein on binding. Circular dichroism studies revealed that the binding led to significant loss in the helicity of hemoglobin. The esterase activity assay further complemented the circular dichroism data. Microcalorimetric study using isothermal titration calorimetry revealed the binding to be exothermic and driven largely by positive entropic contribution. Dissection of the Gibbs energy change proposed the protein-dye complexation to be dominated by non-polyelectrolytic forces. Negative heat capacity change also corroborated the involvement of hydrophobic forces in the binding process. Copyright © 2016 Elsevier B.V. All rights reserved.

Probing Conformational Stability and Dynamics of Erythroid and Nonerythroid Spectrin: Effects of Urea and Guanidine Hydrochloride

PubMed Central

Patra, Malay; Mukhopadhyay, Chaitali; Chakrabarti, Abhijit

2015-01-01

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGu H 2 0) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from. PMID:25617632

UV fluorescence excitation spectroscopy as a non-invasive predictor of epidermal proliferation and clinical performance of cosmetic formulations

NASA Astrophysics Data System (ADS)

Maidhof, Robert; Liebel, Frank; Hwang, Cheng; Ruvolo, Eduardo; Lyga, John

2017-02-01

The epidermis is the outermost layer of skin and is composed of cells primarily containing keratin. It consists of about ten layers of living cells (keratinocytes) and ten layers of dead cells (corneocytes). These cells are continually shed from the outside and replaced from the inside in a process called desquamation which is controlled by two biological events - proliferation and differentiation. One method to non-invasively study biological changes in the skin is using fluorescence excitation spectroscopy. Several characteristic excitation-emission peaks occur in skin that have been related to the epidermal and dermal composition. The magnitude of the peak that occurs at 295nm excitation (F295) has been linked to changes in skin proliferation, cell turnover, epidermal thickening, and skin aging. We hypothesize that changes in this fluorescent signal could be used to assess the potential activity of cosmetic anti-aging compounds to deliver a benefit to skin. Previous work with retinol and glycolic acid, two commonly used actives that effect epidermal proliferation and exfoliation, has demonstrated an increase in F295 (attributed to tryptophan excitation fluorescence). In this study we present the results of a placebo controlled study that aims to correlate changes in F295 with biological performance (epidermal thickening and Ki67 expression).

Online fluorescence spectroscopy for the real-time evaluation of the microbial quality of drinking water.

PubMed

Sorensen, J P R; Vivanco, A; Ascott, M J; Gooddy, D C; Lapworth, D J; Read, D S; Rushworth, C M; Bucknall, J; Herbert, K; Karapanos, I; Gumm, L P; Taylor, R G

2018-06-15

We assessed the utility of online fluorescence spectroscopy for the real-time evaluation of the microbial quality of untreated drinking water. Online fluorimeters were installed on the raw water intake at four groundwater-derived UK public water supplies alongside existing turbidity sensors that are used to forewarn of the presence of microbial contamination in the water industry. The fluorimeters targeted fluorescent dissolved organic matter (DOM) peaks at excitation/emission wavelengths of 280/365 nm (tryptophan-like fluorescence, TLF) and 280/450 nm (humic-like fluorescence, HLF). Discrete samples were collected for Escherichia coli, total bacterial cell counts by flow cytometry, and laboratory-based fluorescence and absorbance. Both TLF and HLF were strongly correlated with E. coli (ρ = 0.71-0.77) and total bacterial cell concentrations (ρ = 0.73-0.76), whereas the correlations between turbidity and E. coli (ρ = 0.48) and total bacterial cell counts (ρ = 0.40) were much weaker. No clear TLF peak was observed at the sites and all apparent TLF was considered to be optical bleed-through from the neighbouring HLF peak. Therefore, a HLF fluorimeter alone would be sufficient to evaluate the microbial water quality at these sources. Fluorescent DOM was also influenced by site operations such as pump start-up and the precipitation of cations on the sensor windows. Online fluorescent DOM sensors are a better indicator of the microbial quality of untreated drinking water than turbidity and they have wide-ranging potential applications within the water industry. Copyright © 2018 British Geological Survey, a component institute of NERC - 'BGS © NERC 2018'. Published by Elsevier Ltd.. All rights reserved.

Resolving the variability of CDOM fluorescence to differentiate the sources and fate of DOM in Lake Taihu and its tributaries.

PubMed

Yao, Xin; Zhang, Yunlin; Zhu, Guangwei; Qin, Boqiang; Feng, Longqing; Cai, Linlin; Gao, Guang

2011-01-01

Taihu Basin is the most developed area in China, which economic development has resulted in pollutants being produced and discharged into rivers and the lake. Lake Taihu is located in the center of the basin, which is characterized by a complex network of rivers and channels. To assess the sources and fate of dissolved organic matter (DOM) in surface waters, we determined the components and abundance of chromophoric dissolved organic matter (CDOM) within Lake Taihu and 66 of its tributaries, and 22 sites along transects from two main rivers. In Lake Taihu, there was a relative less spatial variation in CDOM absorption a(CDOM)(355) with a mean of 2.46 ± 0.69 m⁻¹ compared to the mean of 3.36 ± 1.77 m⁻¹ in the rivers. Two autochthonous tryptophan-like components (C1 and C5), two humic-like components (C2 and C3), and one autochthonous tyrosine-like component (C4) were identified using the parallel factor analysis (PARAFAC) model. The C2 and C3 had a direct relationship with a(CDOM)(355), dissolved organic carbon (DOC), and chemical oxygen demand (COD). The separation of lake samples from river samples, on both axes of the Principal Component Analysis (PCA), showed the difference in DOM fluorophores between these various environments. Components C1 and C5 concurrently showed positive factor 1 loadings, while C4 was close to the negative factor 1 axis. Components C2 and C3 showed positive second factor loadings. The major contribution of autochthonous tryptophan-like components to lake samples is due to the autochthonous production of CDOM in the lake ecosystems. The results also showed that the differences in geology and associated land use control CDOM dynamics, such as the high levels of CDOM with terrestrial characteristics in the northwestern upstream rivers and low levels of CDOM with increased microbial characteristics in the southwestern upstream rivers. Most of river samples from the downstream regions in the eastern and southeastern plains had a similar relative abundance of humic-like fluorescence, with less of the tryptophan-like and more of the tyrosine-like contributions than did samples from upstream regions. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effects on plasma and brain tryptophan in the rat of drugs and hormones that influence the concentration of unesterified fatty acid in the plasma

PubMed Central

Curzon, G.; Knott, P.J.

1974-01-01

1 The effects on tryptophan distribution and metabolism of drugs altering plasma unesterified fatty acid (UFA) concentration were investigated in the rat. 2 UFA and plasma free (i.e. ultrafilterable) tryptophan altered in the same direction. 3 Catecholamines and L-DOPA increased both plasma UFA and free tryptophan. L-DOPA also increased brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) but decreased brain 5-hydroxytryptamine (5-HT). 4 Aminophylline increased plasma UFA and free tryptophan and also brain tryptophan, 5-HT and 5-HIAA. Food deprivation had qualitatively similar effects. 5 Insulin decreased plasma UFA and free tryptophan in both fed and food-deprived rats. However, while in fed rats these changes were associated with small decreases of brain indoles, in food-deprived animals small increases occurred. 6 Nicotinic acid had only small effects in fed rats but it opposed both the UFA and indole changes in food-deprived animals. Total plasma tryptophan increased in nicotinic acid treated, food-deprived rats. 7 There was a tendency towards inverse relations between changes of plasma free and total tryptophan. 8 The results suggest that drugs which influence plasma UFA through actions on cyclic AMP thereby alter the binding of tryptophan to plasma protein and that this leads to altered distribution and metabolism of tryptophan. PMID:4371899

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid 1

PubMed Central

Baldi, Bruce G.; Maher, Barbara R.; Slovin, Janet Pernise; Cohen, Jerry D.

1991-01-01

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. PMID:16668112

Preparation of Curcumin-Piperazine Coamorphous Phase and Fluorescence Spectroscopic and Density Functional Theory Simulation Studies on the Interaction with Bovine Serum Albumin.

PubMed

Pang, Wenzhe; Lv, Jie; Du, Shuang; Wang, Jiaojiao; Wang, Jing; Zeng, Yanli

2017-09-05

In the present study, a new coamorphous phase (CAP) of bioactive herbal ingredient curcumin (CUR) with high solubilitythe was screened with pharmaceutically acceptable coformers. Besides, to provide basic information for the best practice of physiological and pharmaceutical preparations of CUR-based CAP, the interaction between CUR-based CAP and bovine serum albumin (BSA) was studied at the molecular level in this paper. CAP of CUR and piperazine with molar ratio of 1:2 was prepared by EtOH-assisted grinding. The as-prepared CAP was characterized by powder X-ray diffraction, modulated temperature differential scanning calorimetry, thermogravimetric analysis, Fourier-transform infrared, and solid-state 13 C nuclear magnetic resonance. The 1:2 CAP stoichioimetry was sustained by C═O···H hydrogen bonds between the N-H group of the piperazine and the C═O group of CUR; piperazine stabilized the diketo structure of CUR in CAP. The dissolution rate of CUR-piperazine CAP in 30% ethanol-water was faster than that of CUR; the t 50 values were 243.1 min for CUR and 4.378 min for CAP. Furthermore, interactions of CUR and CUR-piperazine CAP with BSA were investigated by fluorescence spectroscopy and density functional theory (DFT) calculation. The binding constants (K b ) of CUR and CUR-piperazine CAP with BSA were 10.0 and 9.1 × 10 3 L mol -1 at 298 K, respectively. Moreover, DFT simulation indicated that the interaction energy values of hydrogen-bonded interaction in the tryptophan-CUR and tryptophan-CUR-piperazine complex were -26.1 and -17.9 kJ mol -1 , respectively. In a conclusion, after formation of CUR-piperazine CAP, the interaction forces between CUR and BSA became weaker.

Deciphering Structural Intermediates and Genotoxic Fibrillar Aggregates of Albumins: A Molecular Mechanism Underlying for Degenerative Diseases

PubMed Central

Naeem, Aabgeena; Amani, Samreen

2013-01-01

The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation. PMID:23342075

[Interaction of protein with charged colloidal particles].

PubMed

Durdenko, E V; Kuznetsova, S M; Basova, L V; Tikhonenko, S A; Saburova, E A

2011-01-01

The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of alpha-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.

Ultrafast Hydration Dynamics and Coupled Water-Protein Fluctuations in Apomyoglobin

NASA Astrophysics Data System (ADS)

Yang, Yi; Zhang, Luyuan; Wang, Lijuan; Zhong, Dongping

2009-06-01

Protein hydration dynamics are of fundamental importance to its structure and function. Here, we characterize the global solvation dynamics and anisotropy dynamics around the apomyoglobin surface in different conformational states (native and molten globule) by measuring the Stokes shift and anisotropy decay of tryptophan with femtosecond-resolved fluorescence upconversion. With site-directed mutagenesis, we designed sixteen mutants with one tryptophan in each, and placed the probe at a desirable position ranging from buried in the protein core to fully solvent-exposed on the protein surface. In all protein sites studied, two distinct solvation relaxations (1-8 ps and 20-200 ps) were observed, reflecting the initial collective water relaxation and subsequent hydrogen-bond network restructuring, respectively, and both are strongly correlated with protein's local structures and chemical properties. The hydration dynamics of the mutants in molten globule state are faster than those observed in native state, indicating that the protein becomes more flexible and less structured when its conformation is converted from fully-folded native state to partially-folded molten globule state. Complementary, fluorescence anisotropy dynamics of all mutants in native state show an increasing trend of wobbling times (40-260 ps) when the location of the probe is changed from a loop, to a lateral helix, and then, to the compact protein core. Such an increase in wobbling times is related to the local protein structural rigidity, which relates the interaction of water with side chains. The ultrafast hydration dynamics and related side-chain motion around the protein surface unravel the coupled water-protein fluctuations on the picosecond time scales and indicate that the local protein motions are slaved by hydrating water fluctuations.

Peptide stabilized amphotericin B nanodisks

PubMed Central

Tufteland, Megan; Pesavento, Joseph B.; Bermingham, Rachelle L.; Hoeprich, Paul D.; Ryan, Robert O.

2007-01-01

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 104. Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND. PMID:17293004

[Spectral Analysis of Dissolved Organic Matter of Tannery Wastewater in the Treatment Process].

PubMed

Fan, Chun-hui; Zhang, Ying-chao; Du, Bo; Song, Juan; Huai, Cui-qian; Wang, Jia-hong

2015-06-01

Tannery industry is one of the major traditional industries and important wastewater sources in China. The existing research mainly focus on the quality of inlet and outlet water, rather than the purification and transformation behavior of dissolved organic matter (DOM) in the treatment process of tannery wastewater. The UV spectra and fluorescence spectroscopy were used to detect the spectral characteristics of water samples in the treatment process, and it is analyzed that the formation process and the linear relationships between total fluorescence intensity and parameters. The results showed: the UV absorbance of DOM in wastewater increased firstly and then decreased with longer wavelength, and the wave peaks were found around the wavelength of 230 nr. The values of A253 /A203 and SUVA254 increased firstly and then decreased, indicating the complex reaction process related to free substituent and aromatic rings. The fluorescence peaks appeared at the regions of λ(ex/em) = 320-350/440- 460 and λ(ex/em) = 270-300/390-420, referred as visible humic-like and visible fulvic-like fluorescence, respectively. With the treatment process of tannery wastewater, the following fluorescence phenomenon were monitored, such as the blue-shift of humic-like fluorescence peak in the hydrolytic acidification tank, the appearance of tryptophan fluorescence peak in the second biochemical pond (λ(ex/em) = 290/340), the weak fluorescence peak in the fourth biochemical pond (λ(ex/em) = 350/520) and the stabilized fluorescence characteristics in the secondary sedimentation tank and water outlet. The achievements are helpful to investigate the degradation and formation behavior of water components, and significant for the fluorescence variation analysis in the treatment system. The removal rate of total fluorescence intensity of tannery wastewater fit better the removal rate of TOC with coefficient of r 0.835 5. The UV spectra and 3D-EEMs are effective to reveal the purification behavior and mechanism of tannery wastewater.

Tryptophan and kynurenine determination in human hair by liquid chromatography.

PubMed

Dario, Michelli F; Freire, Thamires Batello; Pinto, Claudinéia Aparecida Sales de Oliveira; Prado, María Segunda Aurora; Baby, André R; Velasco, Maria Valéria R

2017-10-15

Tryptophan, an amino acid found in hair proteinaceous structure is used as a marker of hair photodegradation. Also, protein loss caused by several chemical/physical treatments can be inferred by tryptophan quantification. Kynurenine is a photo-oxidation product of tryptophan, expected to be detected when hair is exposed mainly to UVB (290-320nm) radiation range. Tryptophan from hair is usually quantified directly as a solid or after alkaline hydrolysis, spectrofluorimetrically. However, these types of measure are not sufficiently specific and present several interfering substances. Thus, this work aimed to propose a quantification method for both tryptophan and kynurenine in hair samples, after alkali hydrolysis process, by using high-performance liquid chromatography (HPLC) with fluorimetric and UV detection. The tryptophan and kynurenine quantification method was developed and validated. Black, white, bleached and dyed (blond and auburn) hair tresses were used in this study. Tryptophan and kynurenine were separated within ∼9min by HPLC. Both black and white virgin hair samples presented similar concentrations of tryptophan, while bleaching caused a reduction in the tryptophan content as well as dyeing process. Unexpectedly, UV/vis radiation did not promote significantly the conversion of tryptophan into its photo-oxidation product and consequently, kynurenine was not detected. Thus, this works presented an acceptable method for quantification of tryptophan and its photooxidation metabolite kynurenine in hair samples. Also, the results indicated that bleaching and dyeing processes promoted protein/amino acids loss but tryptophan is not extensively degraded in human hair by solar radiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Source analysis of organic matter in swine wastewater after anaerobic digestion with EEM-PARAFAC.

PubMed

Zeng, Zhuo; Zheng, Ping; Ding, Aqiang; Zhang, Meng; Abbas, Ghulam; Li, Wei

2017-03-01

Swine wastewater is one of the most serious pollution sources, and it has attracted a great public concern in China. Anaerobic digestion technology is extensively used in swine wastewater treatment. However, the anaerobic digestion effluents are difficult to meet the discharge standard. The results from batch experiments showed that plenty of refractory organic matter remained in the effluents after mesophilic anaerobic digestion for 30 days. The effluent total COD (tCOD) and soluble COD (sCOD) were 483 and 324 mg/L, respectively, with the sCOD/tCOD ratio of 0.671. Fluorescence excitation-emission matrix (EEM) coupled with parallel factor analysis (PARAFAC) revealed that the dissolved organic matter in the effluents was tryptophan-like substance, humic acid substance, and fulvic acid substance. Based on the appearance time during anaerobic digestion, tryptophan-like substance and humic acid substance were inferred to originate from the raw swine wastewater, and the fulvic acid substance was inferred to be formed in the anaerobic digestion. This work has revealed the source of residual organic matter in anaerobic digestion of swine wastewater and has provided some valuable information for the post-treatment.

Tryptophan depletion in context of the inflammatory and general nutritional status of a low-income South African HIV-infected population.

PubMed

Bipath, Priyesh; Levay, Peter F; Viljoen, Margaretha

2016-02-17

The essential amino acid tryptophan cannot be synthesised in the body and must be acquired through dietary intake. Oxidation of tryptophan, due to immune induction of the enzyme indoleamine 2,3-dioxygenase (IDO), is considered to be the main cause of tryptophan depletion in HIV infection and AIDS. We examined plasma tryptophan levels in a low-income sub-Saharan HIV-infected population and compared it to that of developed countries. Tryptophan levels were further examined in context of the general nutritional and inflammatory status. This cross-sectional study included 105 HIV-positive patients recruited from the Kalafong Hospital in Pretoria, South Africa, and 60 HIV-negative controls. Patient tryptophan levels were in general markedly lower than those reported for developed countries. In contrast to reports from developed countries that showed tryptophan levels on average to be 18.8 % lower than their control values, tryptophan levels in our study were 44.1 % lower than our controls (24.4 ± 4.1 vs. 43.6 ± 11.9 μmol/l; p < 0.001). Tryptophan levels correlated with both CD4 counts (r = 0.341; p = 0.004) and with pro-inflammatory activity as indicated by neopterin levels (r = -0.399; p = 0.0001). Nutritional indicators such as albumin and haemoglobin correlated positively with tryptophan and negatively with the pro-inflammatory indicators neopterin, interleukin 6 and C-reactive protein. The most probable causes of the lower tryptophan levels seen in our population are food insecurity and higher levels of inflammatory activity. We contend that inflammation-induced tryptophan depletion forms part of a much wider effect of pro-inflammatory activity on the nutritional profile of HIV-infected patients.

Förster resonance energy transfer between pyrene and bovine serum albumin: effect of the hydrophobic pockets of cyclodextrins.

PubMed

Maity, Arnab; Mukherjee, Puspal; Das, Tarasankar; Ghosh, Prasun; Purkayastha, Pradipta

2012-06-15

The phenomenon of Förster resonance energy transfer (FRET) between pyrene and bovine serum albumin (BSA) protein in presence of cyclodextrins (CDs) is explored in the present work. CDs provide hydrophobic environment and thus the aromatic molecules get encapsulated in them depending on the relative size and space. In this work we revealed that along with pyrene monomer, the side chains of amino acids in BSA can get trapped partly in the hydrophobic cavities of CDs if space permits. While being encapsulated by β-CD as pyrene monomer, it can interact with the BSA tryptophan moiety exposed toward the aqueous environment to form a dimer through π-π interaction. This, in turn, affects the energy transfer process by reducing the efficiency. On the other hand, pyrene excimer gets encapsulated in a γ-CD molecule due to availability of enough space. The excimer shows a new band at a higher wavelength. This further reduces FRET efficiency due to scarcity of acceptor for the tryptophan moieties in BSA. Copyright © 2012 Elsevier B.V. All rights reserved.

Tryptophan biosynthetic enzymes of Staphylococcus aureus.

PubMed

Proctor, A R; Kloos, W E

1973-04-01

Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

Fluorescence Characterization of Clinically-Important Bacteria

PubMed Central

Dartnell, Lewis R.; Roberts, Tom A.; Moore, Ginny; Ward, John M.; Muller, Jan-Peter

2013-01-01

Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems of background fluorescence masking that of bacteria, but in the case of the microfiber cleaning cloth, imaging techniques could morphologically distinguish between stray strands and bacterial contamination. PMID:24098687

Fluorescence characterization of clinically-important bacteria.

PubMed

Dartnell, Lewis R; Roberts, Tom A; Moore, Ginny; Ward, John M; Muller, Jan-Peter

2013-01-01

Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems of background fluorescence masking that of bacteria, but in the case of the microfiber cleaning cloth, imaging techniques could morphologically distinguish between stray strands and bacterial contamination.

Constrained dynamics of the sole tryptophan in the third intracellular loop of the serotonin1A receptor.

PubMed

Pal, Sreetama; Aute, Ramdas; Sarkar, Parijat; Bose, Shroddha; Deshmukh, Mandar V; Chattopadhyay, Amitabha

2018-06-01

G protein-coupled receptors (GPCRs) are major signaling proteins in eukaryotic cells and are important drug targets. In spite of their role in GPCR function, the extramembranous regions of GPCRs are relatively less appreciated. The third intracellular loop (ICL3), which connects transmembrane helices V and VI, is important in this context since its crucial role in signaling has been documented for a number of GPCRs. Unfortunately, the structure of this loop is generally not visualized in x-ray crystallographic studies since this flexible loop is either stabilized using a monoclonal antibody or replaced with lysozyme. In this work, we expressed and purified the ICL3 region of the serotonin 1A receptor and monitored its motional restriction and organization utilizing red edge excitation shift (REES) of its sole tryptophan and circular dichroism (CD) spectroscopy. Our results show that the tryptophan in ICL3 exhibits REES of 4 nm, implying that it is localized in a restricted microenvironment. These results are further supported by wavelength-selective changes in fluorescence anisotropy and lifetime. This constrained dynamics was relaxed upon denaturation of the peptide, thereby suggesting the involvement of the peptide secondary structure in the observed motional restriction, as evident from CD spectroscopy and apparent rotational correlation time. To the best of our knowledge, these results constitute one of the first measurements of motional constraint in the ICL3 region of GPCRs. Our results are relevant in the context of the reported intrinsically disordered nature of ICL3 and its role in providing functional diversity to GPCRs due to conformational plasticity. Copyright © 2018 Elsevier B.V. All rights reserved.

Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection.

PubMed

Kema, I P; Meijer, W G; Meiborg, G; Ooms, B; Willemse, P H; de Vries, E G

2001-10-01

Profiling of the plasma indoles tryptophan, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) is useful in the diagnosis and follow-up of patients with carcinoid tumors. We describe an automated method for the profiling of these indoles in protein-containing matrices as well as the plasma indole concentrations in healthy controls and patients with carcinoid tumors. Plasma, cerebrospinal fluid, and tissue homogenates were prepurified by automated on-line solid-phase extraction (SPE) in Hysphere Resin SH SPE cartridges containing strong hydrophobic polystyrene resin. Analytes were eluted from the SPE cartridge by column switching. Subsequent separation and detection were performed by reversed-phase HPLC combined with fluorometric detection in a total cycle time of 20 min. We obtained samples from 14 healthy controls and 17 patients with metastasized midgut carcinoid tumors for plasma indole analysis. In the patient group, urinary excretion of 5-HIAA and serotonin was compared with concentrations of plasma indoles. Within- and between-series CVs for indoles in platelet-rich plasma were 0.6-6.2% and 3.7-12%, respectively. Results for platelet-rich plasma serotonin compared favorably with those obtained by single-component analysis. Plasma 5-HIAA, but not 5-HTP was detectable in 8 of 17 patients with carcinoid tumors. In the patient group, platelet-rich plasma total tryptophan correlated negatively with platelet-rich plasma serotonin (P = 0.021; r = -0.56), urinary 5-HIAA (P = 0.003; r = -0.68), and urinary serotonin (P <0.0001; r = -0.80). The present chromatographic approach reduces analytical variation and time needed for analysis and gives more detailed information about metabolic deviations in indole metabolism than do manual, single-component analyses.

In situ tryptophan-like fluorometers: assessing turbidity and temperature effects for freshwater applications.

PubMed

Khamis, K; Sorensen, J P R; Bradley, C; Hannah, D M; Lapworth, D J; Stevens, R

2015-04-01

Tryptophan-like fluorescence (TLF) is an indicator of human influence on water quality as TLF peaks are associated with the input of labile organic carbon (e.g. sewage or farm waste) and its microbial breakdown. Hence, real-time measurement of TLF could be particularly useful for monitoring water quality at a higher temporal resolution than available hitherto. However, current understanding of TLF quenching/interference is limited for field deployable sensors. We present results from a rigorous test of two commercially available submersible tryptophan fluorometers (ex ∼ 285, em ∼ 350). Temperature quenching and turbidity interference were quantified in the laboratory and compensation algorithms developed. Field trials were then undertaken involving: (i) an extended deployment (28 days) in a small urban stream; and, (ii) depth profiling of an urban multi-level borehole. TLF was inversely related to water temperature (regression slope range: -1.57 to -2.50). Sediment particle size was identified as an important control on the turbidity specific TLF response, with signal amplification apparent <150 NTU for clay particles and <650 NTU for silt particles. Signal attenuation was only observed >200 NTU for clay particles. Compensation algorithms significantly improved agreement between in situ and laboratory readings for baseflow and storm conditions in the stream. For the groundwater trial, there was an excellent agreement between laboratory and raw in situ TLF; temperature compensation provided only a marginal improvement, and turbidity corrections were unnecessary. These findings highlight the potential utility of real time TLF monitoring for a range of environmental applications (e.g. tracing polluting sources and monitoring groundwater contamination). However, in situations where high/variable suspended sediment loads or rapid changes in temperature are anticipated concurrent monitoring of turbidity and temperature is required and site specific calibration is recommended for long term, surface water monitoring.

Conformational changes in human Hsp70 induced by high hydrostatic pressure produce oligomers with ATPase activity but without chaperone activity.

PubMed

Araujo, Thaís L S; Borges, Julio Cesar; Ramos, Carlos H; Meyer-Fernandes, José Roberto; Oliveira Júnior, Reinaldo S; Pascutti, Pedro G; Foguel, Debora; Palhano, Fernando L

2014-05-13

We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.

Reconstitution of the Hepatic Asialoglycoprotein Receptor with Phospholipid Vesicles

NASA Astrophysics Data System (ADS)

Klausner, Richard D.; Bridges, Kenneth; Tsunoo, Hajime; Blumenthal, Robert; Weinstein, John N.; Ashwell, Gilbert

1980-09-01

A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles. This process is independent of the phase transition of the lipid and effectively restores the specific binding activity of the receptor protein. The insensitivity of the resulting lipid-protein complex to ionic strength provides evidence for a hydrophobic interaction. There is a perturbation of the lipid phase transition concomitant with addition of the protein. Circular dichroism studies indicate that the protein undergoes a conformational change on association with lipid. Binding of specific ligand produces further physical changes in the receptor as indicated by alterations in the tryptophan fluorescence quenching pattern.

Verifying the competition between haloperidol and biperiden in serum albumin through a model based on spectrofluorimetry

NASA Astrophysics Data System (ADS)

Muniz da Silva Fragoso, Viviane; Patrícia de Morais e Coura, Carla; Paulino, Erica Tex; Valdez, Ethel Celene Narvaez; Silva, Dilson; Cortez, Celia Martins

2017-11-01

The aim of this work was to apply mathematical-computational modeling to study the interactions of haloperidol (HLP) and biperiden (BPD) with human (HSA) and bovine (BSA) serum albumin in order to verify the competition of these drugs for binding sites in HSA, using intrinsic tryptophan fluorescence quenching data. The association constants estimated for HPD-HSA was 2.17(±0.05) × 107 M-1, BPD-HSA was 2.01(±0.03) × 108 M-1 at 37 °C. Results have shown that drugs do not compete for the same binding sites in albumin.

Application of portable online LED UV fluorescence sensor to predict the degradation of dissolved organic matter and trace organic contaminants during ozonation.

PubMed

Li, Wen-Tao; Majewsky, Marius; Abbt-Braun, Gudrun; Horn, Harald; Jin, Jing; Li, Qiang; Zhou, Qing; Li, Ai-Min

2016-09-15

This work aims to correlate signals of LED UV/fluorescence sensor with the degradation of dissolved organic matter (DOM) and trace-level organic contaminants (TOrCs) during ozonation process. Six sets of bench-scale ozonation kinetic experiments incorporated with three different water matrices and 14 TOrCs of different reactivity (group I ∼ V) were conducted. Calibrated by tryptophan and humic substances standards and verified by the lab benchtop spectroscopy, the newly developed portable/online LED sensor, which measures the UV280 absorbance, protein-like and humic-like fluorescence simultaneously, was feasible to monitor chromophores and fluorophores with good sensitivity and accuracy. The liquid chromatography with organic carbon detector combined with 2D synchronous correlation analysis further demonstrated how the DOM components of large molecular weight were transformed into small moieties as a function of the decrease of humic-like fluorescence. For TOrCs, their removal rates were well correlated with the decrease of the LED UV/fluorescence signals, and their elimination patterns were mainly determined by their reactivity with O3 and hydroxyl radicals. At approximately 50% reduction of humic-like fluorescence almost complete oxidation of TOrCs of group I and II was reached, a similar removal percentage (25-75%) of TOrCs of group III and IV, and a poor removal percentage (<25%) of group V. This study might contribute to the smart control of advanced oxidation processes for the water and wastewater treatment in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of different tryptophan sources on amino acids availability to the brain and mood in healthy volunteers.

PubMed

Markus, C Rob; Firk, Christine; Gerhardt, Cindy; Kloek, Joris; Smolders, Gertjan F

2008-11-01

Reduced brain serotonin function is acknowledged as a vulnerability factor for affective disturbances. Since the production of serotonin is limited by the availability of its plasma dietary amino acid precursor tryptophan (TRP), the beneficial effects of tryptophan-rich alpha-lactalbumin whey protein (ALAC) have recently been studied. The effects of ALAC remain rather modest, and alternative protein sources of tryptophan may be more effective. We tested whether hydrolyzed protein (HPROT) has greater effects on the plasma TRP/large neutral amino acids (LNAA) ratio and mood than intact ALAC protein in healthy volunteers. In a double-blind, randomized cross-over study, plasma amino acids and mood were repeatedly measured in 18 healthy subjects before and after intake of ALAC and HPROT as well as after placebo protein, pure tryptophan, and a tryptophan-containing synthetic peptide. Except for the placebo protein, all interventions contained 0.8 g TRP. Significantly faster and greater increases in plasma TRP/LNAA were found after HPROT than after ALAC. In addition, the effects of HPROT on plasma TRP/LNAA were comparable with the effects of the tryptophan-containing synthetic peptide and even exceeded the effect of pure tryptophan. Sixty minutes after intake, mood was improved only following intake of HPROT and pure tryptophan, whereas longer-lasting mood effects were only found after intake of HPROT. The use of a tryptophan-rich hydrolyzed protein source may be more adequate to increase brain tryptophan and 5-HT function compared with intact alpha-lactalbumin protein or pure tryptophan.

Switch-on fluorescent/FRET probes to study human histidine triad nucleotide binding protein 1 (hHint1), a novel target for opioid tolerance and neuropathic pain.

PubMed

Shah, Rachit; Zhou, Andrew; Wagner, Carston R

2017-12-13

Histidine Triad Nucleotide Binding Protein 1 (Hint1) has emerged to be an important post-synaptic protein associated with a variety of central nervous system disorders such as pain, addiction, and schizophrenia. Recently, inhibition of histidine nucleotide binding protein 1 (Hint1) with a small nucleoside inhibitor has shown promise as a new therapeutic strategy for the treatment of neuropathic pain. Herein, we describe the first rationally designed small molecule switch-on probes with dual fluorescence and FRET properties to study Hint1. Two non-natural fluorescent nucleosides with a fluorescent lifetime of 20 and 25 ns were each coupled through a linker to the indole ring, i.e. probes 7 and 8. Both probes were found to be water soluble and quenched intramolecularly via photoinduced electron transfer (PET) resulting in minimal background fluorescence. Upon incubating with Hint1, compound 7 and 8 exhibited a 40- and 16-fold increase in the fluorescence intensity compared to the control. Compounds 7 and 8 bind Hint1 with a dissociation constant of 0.121 ± 0.02 and 2.2 ± 0.36 μM, respectively. We demonstrate that probe 8 exhibits a switch-on FRET property with an active site tryptophan residue (W123). We show the utility of probes in performing quantitative ligand displacement studies, as well as in selective detection of Hint1 in the cell lysates. These probes should be useful for studying the dynamics of the active site, as well as for the development of fluorescence lifetime based high throughput screening assay to identify novel inhibitors for Hint1 in future.

Rapid measurement of meat spoilage using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Wu, Binlin; Dahlberg, Kevin; Gao, Xin; Smith, Jason; Bailin, Jacob

2017-02-01

Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature ( 19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer ( -12° C), the changes are much less or even unnoticeable over a-week-long storage.

Biophysical and computational characterization of vandetanib-lysozyme interaction

NASA Astrophysics Data System (ADS)

Kabir, Md. Zahirul; Hamzah, Nur Aziean Binti; Ghani, Hamidah; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad

2018-01-01

Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka = 4.96-3.14 × 103 M-1). Thermodynamic data (ΔS = + 12.82 J mol-1 K-1; ΔH = - 16.73 kJ mol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.

Static and kinetic studies of calmodulin and melittin complex.

PubMed

Itakura, M; Iio, T

1992-08-01

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.

Conformational transition of κ-casein in micellar environment: Insight from the tryptophan fluorescence

NASA Astrophysics Data System (ADS)

Mishra, Smruti; Meher, Geetanjali; Chakraborty, Hirak

2017-11-01

Intrinsically disordered proteins (IDPs) are under intense analysis due to their structural flexibility and importance in biological functions. Minuscule modulation in the microenvironment induces significant conformational changes in IDPs, and these non-native conformations of the IDPs often induce aggregation and cause cell death. Changes in the membrane composition often change the microenvironment, which promote conformational change and aggregation of IDPs. κ-Casein, an important milk protein, belongs to the class of IDPs containing net negative charges. In this present work, we have studied the interaction of κ-casein with cetyltrimethyl ammonium bromide (CTAB), a positively charged surfactant, utilizing various steady state fluorescence, time-resolved fluorescence and circular dichroism spectroscopy. Our results clearly indicate that κ-casein undergoes at least two conformational transitions in presence of various concentrations of CTAB. The intrinsically disordered κ-casein assumes a partially folded conformation at lower concentration of CTAB, which adopts an unstructured conformation at higher concentration of CTAB. The partially folded conformation of κ-casein at a lower CTAB concentration might be induced by the favorable electrostatic interaction between the positively charged surfactant headgroup and net negative charges of the protein, whereas surfactant nature of CTAB is being pronounced at higher concentration of CTAB.

Electric Dipole Transition Moments and Solvent-Dependent Interactions of Fluorescent Boron-Nitrogen Substituted Indole Derivatives.

PubMed

Saif, Mari; Widom, Julia R; Xu, Senmiao; Abbey, Eric R; Liu, Shih-Yuan; Marcus, Andrew H

2015-06-25

Fluorescent analogues of the indole side chain of tryptophan can be useful spectroscopic probes of protein-protein and protein-DNA interactions. Here we present linear dichroism and solvent-dependent spectroscopic studies of two fluorescent analogues of indole, in which the organic C═C unit is substituted with the isosteric inorganic B-N unit. We studied the so-called "external" BN indole, which has C2v symmetry, and the "fused" BN indole with Cs symmetry. We performed a combination of absorption and fluorescence spectroscopy, ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE) films, and quantum chemical calculations on both BN indole compounds. Our measurements allowed us to characterize the degree of alignment for both molecules in stretched PE films. We thus determined the orientations and magnitudes of the two lowest energy electric dipole transition moments (EDTMs) for external BN indole, and the two lowest energy EDTMs for fused BN indole within the 30 000-45 000 cm(-1) spectral range. We compared our experimental results to those of quantum chemical calculations using standard density functional theory (DFT). Our theoretical predictions for the low-energy EDTMs are in good agreement with our experimental data. The absorption and fluorescence spectra of the external and the fused BN indoles are sensitive to solvent polarity. Our results indicate that the fused BN indole experiences much greater solvation interactions with polar solvents than does the external BN indole.

Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

NASA Astrophysics Data System (ADS)

Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

2016-03-01

Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

NASA Astrophysics Data System (ADS)

Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

2014-05-01

Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were performed in pellets (boric and humic acids mixture) using a portable system built by Embrapa Instrumentation. It comprises a diode laser (Coherent - CUBE) emitting at 405 nm (50 mW), and the detection of emission by a high sensitivity mini-spectrometer (USB4000 - Ocean Optics) using a range from 440 to 800 nm. In first step, the 3D tensors were then treated by the CP/PARAFAC algorithm to decompose the signal response after removing the diffusion signal : three components were extracted with a CORCONDIA over 60%. The first component can be associate an artefact of the measurement or boric acid fluorescence, the second and third component could the related to the two different fluorescence contributions of tryptophan molecule, one with central excitation/emission in 290/360 nm and other in 350/465 nm. The presence of a small quantity (i.e. few percent in mass) of humic acid (HA) is quenching drastically the TRP fluorescence. Complementary, measurements will be performed to understand this behaviour taking in account the absorption wavelength by the surface (colour) and by measuring the time life fluorescence of the samples. Humic acid fluorescence in pellets (BA and HA) cannot be observed using lamp + monochromator excitation due to low intensity of source. The same pellets were measure using LIFS system, and fluorescence intensity increased as a function of concentration of HA until occur the inner filter effect from 300 ppm, similar to the behaviour of HA in solution. Even whether solid surface measurements are easier, understanding is not yet clear. More investigation needs to be done. Moreover, it should be important to know if the use of CP/PARAFAC decomposition for such data is relevant with the trilinear model. References Milori, D.M.B.P., Galeti, H.V.A., Martin-Neto, L., Dieckow, J., González-Pérez, M., Bayer, C., Salton, J., 2006. Organic Matter Study of Whole Soil Samples Using Laser-Induced Fluorescence Spectroscopy. Soil Science Society of America Journal 70, 57. N. Senesi, TM, M., MR, P., Brunetti., G., 1991. Characterization, differentiation and classification of humic substances by fluorescence spectroscopy. Soil Science 152, 259-271. Senesi, N., 1990. Molecular and quantitative aspects of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals. Analytica Chimica Acta 232, 77-106.

Tryptophan Metabolism and White Matter Integrity in Schizophrenia

PubMed Central

Chiappelli, Joshua; Postolache, Teodor T; Kochunov, Peter; Rowland, Laura M; Wijtenburg, S Andrea; Shukla, Dinesh K; Tagamets, Malle; Du, Xiaoming; Savransky, Anya; Lowry, Christopher A; Can, Adem; Fuchs, Dietmar; Hong, L Elliot

2016-01-01

Schizophrenia is associated with abnormalities in the structure and functioning of white matter, but the underlying neuropathology is unclear. We hypothesized that increased tryptophan degradation in the kynurenine pathway could be associated with white matter microstructure and biochemistry, potentially contributing to white matter abnormalities in schizophrenia. To test this, fasting plasma samples were obtained from 37 schizophrenia patients and 38 healthy controls and levels of total tryptophan and its metabolite kynurenine were assessed. The ratio of kynurenine to tryptophan was used as an index of tryptophan catabolic activity in this pathway. White matter structure and function were assessed by diffusion tensor imaging (DTI) and 1H magnetic resonance spectroscopy (MRS). Tryptophan levels were significantly lower (p<0.001), and kynurenine/tryptophan ratios were correspondingly higher (p=0.018) in patients compared with controls. In patients, lower plasma tryptophan levels corresponded to lower structural integrity (DTI fractional anisotropy) (r=0.347, p=0.038). In both patients and controls, the kynurenine/tryptophan ratio was inversely correlated with frontal white matter glutamate level (r=−0.391 and −0.350 respectively, p=0.024 and 0.036). These results provide initial evidence implicating abnormal tryptophan/kynurenine pathway activity in changes to white matter integrity and white matter glutamate in schizophrenia. PMID:27143602

Antihypertensive and cardioprotective effects of the dipeptide isoleucine-tryptophan and whey protein hydrolysate.

PubMed

Martin, M; Kopaliani, I; Jannasch, A; Mund, C; Todorov, V; Henle, T; Deussen, A

2015-12-01

Angiotensin-converting enzyme inhibitors are treatment of choice in hypertensive patients. Clinically used inhibitors exhibit a structural similarity to naturally occurring peptides. This study evaluated antihypertensive and cardioprotective effects of ACE-inhibiting peptides derived from food proteins in spontaneously hypertensive rats. Isoleucine-tryptophan (in vitro IC50 for ACE = 0.7 μm), a whey protein hydrolysate containing an augmented fraction of isoleucine-tryptophan, or captopril was given to spontaneously hypertensive rats (n = 60) over 14 weeks. Two further groups, receiving either no supplement (Placebo) or intact whey protein, served as controls. Systolic blood pressure age-dependently increased in the Placebo group, whereas the blood pressure rise was effectively blunted by isoleucine-tryptophan, whey protein hydrolysate and captopril (-42 ± 3, -38 ± 5, -55 ± 4 mm Hg vs. Placebo). At study end, myocardial mass was lower in isoleucine-tryptophan and captopril groups but only partially in the hydrolysate group. Coronary flow reserve (1 μm adenosine) was improved in isoleucine-tryptophan and captopril groups. Plasma ACE activity was significantly decreased in isoleucine-tryptophan, hydrolysate and captopril groups, but in aortic tissue only after isoleucine-tryptophan or captopril treatment. This was associated with lowered expression and activity of matrix metalloproteinase-2. Following isoleucine-tryptophan and captopril treatments, gene expression of renin was significantly increased indicating an active feedback within renin-angiotensin system. Whey protein hydrolysate and isoleucine-tryptophan powerfully inhibit plasma ACE resulting in antihypertensive effects. Moreover, isoleucine-tryptophan blunts tissue ACE activity, reduces matrix metalloproteinase-2 activity and improves coronary flow reserve. Thus, whey protein hydrolysate and particularly isoleucine-tryptophan may serve as innovative food additives with the goal of attenuating hypertension. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Tryptophan promotes morphological and physiological differentiation in Streptomyces coelicolor.

PubMed

Palazzotto, Emilia; Renzone, Giovanni; Fontana, Pietro; Botta, Luigi; Scaloni, Andrea; Puglia, Anna Maria; Gallo, Giuseppe

2015-12-01

The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of the CDA gene cluster regulator cdaR and, coherently, CDA production. Surprisingly, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure. Combined 2D-DIGE and nano liquid chromatography electrospray linear ion trap tandem mass spectrometry (LC-ESI-LIT-MS/MS) analyses revealed that tryptophan exerts a growth-stage-dependent global effect on S. coelicolor proteome, stimulating anabolic pathways and promoting the accumulation of key factors associated with morphological and physiological differentiation at the late growth stages. Phenotypic observations by scanning electron microscopy and spore production assays demonstrated an increased sporulation in the presence of tryptophan. Transcriptional analysis of catabolic genes kynA and kynB suggested that the actinomycete also uses tryptophan as a carbon and nitrogen source. In conclusion, this study originally provides the molecular basis underlying the stimulatory effect of tryptophan on the production of antibiotics and morphological development program of this actinomycete.

Co-treatment of fruit and vegetable waste in sludge digesters: Chemical and spectroscopic investigation by fluorescence and Fourier transform infrared spectroscopy.

PubMed

Provenzano, Maria Rosaria; Cavallo, Ornella; Malerba, Anna Daniela; Di Maria, Francesco; Cucina, Mirko; Massaccesi, Luisa; Gigliotti, Giovanni

2016-04-01

In a previous work co-digestion of food waste and sewage sludge was performed in a pilot apparatus reproducing operating conditions of an existing full scale digester and processing waste mixed sludge (WMS) and fruit and vegetable waste (FVW) at different organic loading rates. An analysis of the relationship among bio-methane generation, process stability and digestate phytotoxicity was conducted. In this paper we considered humification parameters and spectroscopic analysis. Humification parameters indicated a higher not humified fraction (NH) and a lower degree of humification (DH) of FVW with respect to WMS (NH=19.22 and 5.10%; DH=36.65 and 61.94% for FVW and WMS, respectively) associated with their different chemical compositions and with the stabilization process previously undergone by sludge. FVW additions seemed to be favourable from an agronomical point of view since a lower percentage of organic carbon was lost. Fourier transform infrared spectra suggested consumption of aliphatics associated with rising in bio-methane generation followed by accumulation of aliphatics and carboxylic acids when the biogas production dropped. The trend of peaks ratios can be used as an indicator of the process efficiency. Fluorescence intensity of peak B associated with tryptophan-like substances and peak D associated with humic-like substances observed on tridimensional Excitation Emission Matrix maps increased up to sample corresponding to the highest rate of biogas production. Overall spectroscopic results provided evidence of different chemical pathways of anaerobic digestion associated with increasing amount of FVW which led to different levels of biogas production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Relationship between soluble microbial products (SMP) and effluent organic matter (EfOM): characterized by fluorescence excitation emission matrix coupled with parallel factor analysis.

PubMed

Yu, Huarong; Qu, Fangshu; Sun, Lianpeng; Liang, Heng; Han, Zhengshuang; Chang, Haiqing; Shao, Senlin; Li, Guibai

2015-02-01

Effluent organic matter (EfOM) originating from wastewater treatment plant (WWTP) is of significant concern, as it not only influences the discharge quality of WWTP but also exerts a significant effect on the efficiency of the downstream advanced treatment facilities. Soluble microbial products (SMP) is a major part of EfOM. In order to further understand the relationship between soluble microbial products (SMP) and EfOM, and in turn, to propose measures for EfOM control, the formation of SMP and EfOM in identical activated sludge sequencing batch reactors (SBR) with different feed water was investigated using fluorescence excitation and emission spectroscopy matrix coupled with parallel factor analysis (EEM-PARAFAC) as well as other organic matter quantification tools. Results showed that EfOM contained not only SMP but also a considerable amount of allochthonous organic matter that derived not merely from natural organic matter (NOM). Four components in EfOM/SMP were identified by EEM-PARAFAC. Tyrosine-like substances in EfOM (Component 3, λex/em=270/316 nm) were mainly originated from utilization associated products (UAP) of SMP. Tryptophan-like substances (Component 2, λex/em=280/336 nm) as well as fulvic-like and humic-like substances in EfOM (Component 1, λex/em=240(290)/392 nm and Component 4, λex/em=260(365)/444 nm) were majorly derived from the refractory substances introduced along with the influent, among which Component 2 was stemmed from sources other than NOM. As solid retention time (SRT) increased, Component 2 and polysaccharides in SMP/EfOM decreased, while Component 4 in SMP increased. Copyright © 2014 Elsevier Ltd. All rights reserved.

A review of the immunomodulatory role of dietary tryptophan in livestock and poultry.

PubMed

Bai, Miaomiao; Liu, Hongnan; Xu, Kang; Oso, Abimbola Oladele; Wu, Xin; Liu, Gang; Tossou, Myrlene Carine B; Al-Dhabi, Naif Abdullah; Duraipandiyan, Veeramuthu; Xi, Qianyun; Yin, Yinlong

2017-01-01

Tryptophan, a nutritionally essential amino acid, is active in the regulation of immune responses in animals. The products of tryptophan metabolism, such as indoleamine 2,3-dioxygenase, kynurenine, quinolinic acid, and melatonin, may improve immunity in an organism and induce anti-inflammatory responses. The immune tolerance processes mediated by tryptophan metabolites are not well understood. Recent studies have reported that the enzymes that break down tryptophan through the kynurenine metabolic pathway are found in numerous cell types, including immunocytes. Moreover, some tryptophan metabolites have been shown to play a role in the inhibition of T lymphocyte proliferation, elevation of immunoglobulin levels in the blood, and promotion of antigen-presenting organization in tissues. This review summarizes the effects and mechanisms of tryptophan and metabolites in immune functions in livestock and poultry. It also highlights the areas in which our understanding of the role(s) of tryptophan is incomplete and suggests possible future research that might prove of benefit to livestock and poultry producers.

Requirement for tryptophan by milkfish (Chanos chanos Forsskal) juveniles.

PubMed

Coloso, R M; Tiro, L B; Benitez, L V

1992-05-01

Groups of milkfish juveniles (mean initial weight 7.7 g) were fed semipurified diets containing 0.9, 1.4, 2.1, 3.1, 4.1 and 6.1 g tryptophan/kg dry diet for 12 weeks. The mean crude protein content of the diets (containing white fishmeal, gelatin and free amino acid mixture to simulate the pattern of hydrolysed milkfish protein) was 49%. On the basis of the growth response, the tryptophan requirement of milkfish juveniles was estimated to be 3.1 g/kg diet. Fish fed low levels of tryptophan exhibited low weight gains and poor feed conversion ratios. Survival (92-100%) was consistently high in all treatments. Fish fed diets containing tryptophan levels greater than 3.1 g/kg had slightly lower survival rates. The activity of hepatic tryptophan pyrrolase showed no significant differences with increasing dietary tryptophan levels. No nutritional deficiency signs were observed other than the depression in growth rates in fish given the tryptophan deficient diets.

Spectroscopic analyses on sonocatalytic damage to bovine serum albumin (BSA) induced by ZnO/hydroxylapatite (ZnO/HA) composite under ultrasonic irradiation

NASA Astrophysics Data System (ADS)

Wang, Zhiqiu; Li, Ying; Wang, Jun; Zou, Mingming; Gao, Jingqun; Kong, Yumei; Li, Kai; Han, Guangxi

ZnO/hydroxylapatite (ZnO/HA) composite with HA molar content of 6.0% was prepared by the method of precipitation and heat-treated at 500 °C for 40 min and was characterized by powder X-ray diffraction (XRD). The sonocatalytic activities of ZnO/HA composite was carried out through the damage of bovine serum albumin (BSA) in aqueous solution. Furthermore, the effects of several factors on the damage of BSA molecules were evaluated by means of UV-vis and fluorescence spectra. Experimental results indicated that the damage degree of BSA aggravated with the increase of ultrasonic irradiation time, irradiation power and ZnO/HA addition amount, but weakened with the increase of solution acidity and ionic strength. In addition, the damage site to BSA was also studied by synchronous fluorescence technology and the damage site was mainly at tryptophan (Trp) residue. This paper provides a valuable reference for driving sonocatalytic method to treat tumor in clinic application.

Sensing of heavy metal ions by intrinsic TMV coat protein fluorescence.

PubMed

Bayram, Serene S; Green, Philippe; Blum, Amy Szuchmacher

2018-04-15

We propose the use of a cysteine mutant of TMV coat protein as a signal transducer for the selective sensing and quantification of the heavy metal ions, Cd 2+ , Pb 2+ , Zn 2+ and Ni 2+ based on intrinsic tryptophan quenching. TMV coat protein is inexpensive, can be mass-produced since it is expressed and extracted from E-coli. It also displays several different functional groups, enabling a wide repertoire of bioconjugation chemistries; thus it can be easily integrated into functional devices. In addition, TMV-ion interactions have been widely reported and utilized for metallization to generate organic-inorganic hybrid composite novel materials. Building on these previous observations, we herein determine, for the first time, the TMV-ion binding constants assuming the static fluorescence quenching model. We also show that by comparing TMV-ion interactions between native and denatured coat protein, we can distinguish between chemically similar heavy metal ions such as cadmium and zinc ions. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of a pre-active conformation of a pentameric channel receptor

PubMed Central

Menny, Anaïs; Lefebvre, Solène N; Schmidpeter, Philipp AM; Drège, Emmanuelle; Fourati, Zaineb; Delarue, Marc; Edelstein, Stuart J; Nimigean, Crina M; Joseph, Delphine; Corringer, Pierre-Jean

2017-01-01

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation. DOI: http://dx.doi.org/10.7554/eLife.23955.001 PMID:28294942

Effect of Dioxygen on Copper(II) Binding to α-Synuclein

PubMed Central

Lucas, Heather R.; Lee, Jennifer C.

2010-01-01

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (Kd(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M*DV(F/W), where M*= methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a CuII-Trp/Phe (cation-π) interaction. PMID:20064662

Natural organic matter properties in Swedish agricultural streams

NASA Astrophysics Data System (ADS)

Bieroza, Magdalena; Kyllmar, Katarina; Bergström, Lars; Köhler, Stephan

2017-04-01

The following paper shows natural organic matter (NOM) properties of stream water samples collected from 8 agricultural streams and 12 agricultural observational fields in Sweden. The catchments and observational fields cover a broad range of environmental (climate, soil type), land use and water quality (nutrient and concentrations, pH, alkalinity) characteristics. Stream water samples collected every two weeks within an ongoing Swedish Monitoring Programme for Agriculture have been analysed for total/dissolved organic carbon, absorbance and fluorescence spectroscopy. A number of quantitative and qualitative spectroscopic parameters was calculated to help to distinguish between terrestrially-derived, refractory organic material and autochthonous, labile material indicative of biogeochemical transformations of terrestrial NOM and recent biological production. The study provides insights into organic matter properties and carbon budgets in agricultural streams and improves understanding of how agricultural catchments transform natural and anthropogenic fluxes of organic matter and nutrients. The insights from the grab sampling are supported by high-frequency turbidity, fulvic-like and tryptophan-like fluorescence measurements with in situ optical sensor.

Seasonal and downstream alterations of dissolved organic matter and dissolved inorganic ions in a human-impacted mountainous tributary of the Yellow River, China.

PubMed

Zhang, Shurong; Bai, Yijuan; Wen, Xin; Ding, Aizhong; Zhi, Jianhui

2018-04-22

Human activities impose important disturbances on both organic and inorganic chemistry in fluvial systems. In this study, we investigated the intra-annual and downstream variations of dissolved organic carbon (DOC), dissolved organic matter (DOM) excitation-emission matrix fluorescence (EEM) with parallel factor analysis (PARAFAC), major ions, and dissolved inorganic nitrogen (DIN) species in a mountainous tributary of the Yellow River, China. Both DOM quantity and quality, as represented by DOC and DOM fluorescence respectively, changed spatially and seasonally in the studied region. Fluorescence intensity of tryptophan-like components (C3) were found much higher at the populated downstream regions than in the undisturbed forested upstream regions. Seasonally, stronger fluorescence intensity of protein-like components (C3 and C4) was observed in the low-flow period (December) and in the medium-flow period (March) than in the high-flow period (May), particularly for the downstream reaches, reflecting the dominant impacts of wastewater pollution in the downstream regions. In contrast to the protein-like fluorescence, humic-like fluorescence components C1 and C2 exhibited distinctly higher intensity in the high-flow period with smaller spatial variation indicating strong flushing effect of increasing water discharge on terrestrial-sourced humic-like materials in the high-flow period. Pollution-affected dissolved inorganic ions, particularly Na + , Cl - , and NH 4 + -N, showed similar spatial and seasonal variations with protein-like fluorescence of DOM. The significant positive correlations between protein-like fluorescence of DOM and pollution-affected ions, particularly Na + , Cl - , and NH 4 + -N, suggested that there were similar pollution sources and transportation pathways of both inorganic and organic pollutants in the region. The combination of DOM fluorescence properties and inorganic ions could provide an important reference for the pollution source characterization and river basin management.

Absorption and fluorescence properties of chromophoric dissolved organic matter: implications for the monitoring of water quality in a large subtropical reservoir.

PubMed

Liu, Xiaohan; Zhang, Yunlin; Shi, Kun; Zhu, Guangwei; Xu, Hai; Zhu, Mengyuan

2014-12-01

The development of techniques for real-time monitoring of water quality is of great importance for effectively managing inland water resources. In this study, we first analyzed the absorption and fluorescence properties in a large subtropical reservoir and then used a chromophoric dissolved organic matter (CDOM) fluorescence monitoring sensor to predict several water quality parameters including the total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD), dissolved organic carbon (DOC), and CDOM fluorescence parallel factor analysis (PARAFAC) components in the reservoir. The CDOM absorption coefficient at 254 nm (a(254)), the humic-like component (C1), and the tryptophan-like component (C3) decreased significantly along a gradient from the northwest to the lake center, northeast, southwest, and southeast region in the reservoir. However, no significant spatial difference was found for the tyrosine-like component (C2), which contributed only four marked peaks. A highly significant linear correlation was found between the a(254) and CDOM concentration measured using the CDOM fluorescence sensor (r(2) = 0.865, n = 76, p < 0.001), indicating that CDOM concentrations could act as a proxy for the CDOM absorption coefficient measured in the laboratory. Significant correlations were also found between the CDOM concentration and TN, TP, COD, DOC, and the maximum fluorescence intensity of C1, suggesting that the real-time monitoring of CDOM concentrations could be used to predict these water quality parameters and trace the humic-like fluorescence substance in clear aquatic ecosystems with DOC <2 mg/L and total suspended matter (TSM) concentrations <15 mg/L. These results demonstrate that the CDOM fluorescence sensor is a useful tool for on-line water quality monitoring if the empirical relationship between the CDOM concentration measured using the CDOM fluorescence sensor and the water quality parameters is calibrated and validated.

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions. PMID:23284658

Effect of fluorescent particle size on the modulation efficiency of ultrasound-modulated fluorescence.

PubMed

Liu, Yuan; Yuan, Baohong; Vignola, Joseph

2012-01-01

To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20 nm, 200 nm, and 1.0 µm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5 nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5 nm to 1 µm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) were maintained regardless of the fluorescent particle sizes.

Effect of fluorescent particle size on the modulation efficiency of ultrasound-modulated fluorescence

PubMed Central

Liu, Yuan; Yuan, Baohong; Vignola, Joseph

2013-01-01

To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20 nm, 200 nm, and 1.0 µm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5 nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5 nm to 1 µm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) were maintained regardless of the fluorescent particle sizes. PMID:24179476

Stability of Pin1 as revealed by thermal and spectroscopic studies

NASA Astrophysics Data System (ADS)

Wang, Jing-Zhang; Lin, Tao; Zhu, Guo-Fei; Du, Lin-Fang

2010-06-01

Pin1 is a two-domain enzyme which has peptidyl-prolyl cis/trans isomerase activity. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer's disease target. The thermal stability of Pin1 was studied intensively by SDS-PAGE, enzymatic activity assay, intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. The activity of Pin1 gradually decreased above 40 °C, and the Tm was 57.6 ± 1.0 °C. Fluorescence experiments indicated that heat treatment induced changes in the substructures in Pin1, resulting in that the polarity in the microenvironments of the tryptophan residues increased. It is assumed that the thermal denaturation of Pin1 involved a three-state transition. The intermediate state of Pin1 at about 60 °C was confirmed by fluorescence emission spectra, the synchronous fluorescence spectra and CD measurements. Decreases in α-helix and β-sheet appeared above 40 °C, which was balanced by an enhancement in unordered coil. The Tm values calculated from α-helix transition and β-sheet transition were 54.6 ± 0.6 °C and 70.7 ± 3.3 °C, respectively. Our results illustrated that Pin1 had a relatively high thermal stability and the WW domain had a higher stability than the PPIase domain.

Efficient triplet harvesting by fluorescent molecules through exciplexes for high efficiency organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Park, Young-Seo; Kim, Kwon-Hyeon; Kim, Jang-Joo

2013-04-01

Efficient triplet harvesting from exciplexes by reverse intersystem crossing (RISC) is reported using a fluorescent molecular system composed of the 4,4',4″-tris(N-carbazolyl)-triphenylamine and bis-4,6-(3,5-di-3-pyridylphenyl)-2-methylpyrimidine. The exciplex forming material system shows the efficient delayed fluorescence emission. As a result, almost 100% PL efficiency at 35 K and 10% external quantum efficiency at 195 K are achieved from the exciplex. The delayed fluorescence of the exciplex clearly demonstrates that a significant proportion of the triplet exciplexes is harvested through the RISC.

Biotransformation of indole derivatives by mycelial cultures.

PubMed

Alarcón, Julio; Cid, Eliseo; Lillo, Luis; Céspedesa, Carlos; Aguila, Sergio; Alderete, Joel B

2008-01-01

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.

Improving the quality of infant sleep through the inclusion at supper of cereals enriched with tryptophan, adenosine-5'-phosphate, and uridine-5'-phosphate.

PubMed

Cubero, Javier; Chanclón, Belen; Sánchez, Soledad; Rivero, Montserrat; Rodríguez, Ana Beatriz; Barriga, Carmen

2009-12-01

The present study evaluated whether the administration of cereals enriched with nutrients that are facilitators of sleep could help improve the sleep of infants who had sleep disorders at night time. Thirty infants aged 8-16 months with sleep disorders involving at least three nocturnal waking episodes took part in the study. They were given a night-time 'sleep facilitating cereal' product containing 225 mg tryptophan, 5.3 mg adenosine-5'-P, and 6.3 mg uridine-5'-P per 100 g of product. These cereals were given in a double-blind procedure lasting 5 weeks, with ingestion of the cereal between 18:00 and 06:00. In the control week, the children received a standard cereal (75 mg tryptophan/100 g product without nucleotides) dissolved in a standard formula milk (231.5 mg tryptophan, 2.6 mg adenosine-5'-P, 5 mg uridine-5'-P, per 100 g product). In one experimental week, the children received the night-time sleep facilitating cereal together with the standard formula milk. In another week, they received the sleep facilitating cereal together with a night milk specially formulated to attain the sleep rhythm (480 mg tryptophan, 8.8 mg uridine-5'-P, and 7.6 mg adenosine-5'-P per 100 g product). The three experimental weeks were separated by two wash-out weeks in which the milk and cereal administered was identical in composition to that of the control week. All the infants received a programmed writer actimeter which they wore continually, attached to their ankles, to record their motor activity. The recorded activity was used to calculate information about the time in bed, assumed sleep, actual sleep, sleep efficiency, sleep latency, immobility, and total activity. The infants receiving the enriched cereal during the time of darkness showed improvements in their sleep parameters, regardless of whether the milk they took at night was standard or enriched with tryptophan, adenosine-5'-P, and uridine-5'-P. In summary, the administration of enriched cereals led to an improvement in sleep, regardless of the type of infant milk used. These results support the concept of chrononutrition since they confirm that the sleep/wake rhythm can be influenced by diet.

Molecular Cloning and Analysis of the Tryptophan oxygenase Gene in the Silkworm, Bombyx mori

PubMed Central

Yan, Liu; Zhi-Qi, Meng; Bao-Long, Niu; Li-Hua, He; Hong-Biao, Weng; Wei-Feng, Shen

2008-01-01

A Bombyx mori L. (Lepidoptera: Bombycidae) gene encoding tryptophan oxygenase has been molecularly cloned and analyzed. The tryptophan oxygenase cDNA had 1374 nucleotides that encoded a 401 amino acid protein with an estimated molecular mass of 46.47 kDa and a PI of 5.88. RT-PCR analysis showed that the B. mori tryptophan oxygenase gene was transcribed in all examined stages. Tryptophan oxygenase proteins are relatively well conserved among different orders of arthropods. PMID:20331401

Dietary tryptophan intake and suicide rate in industrialized nations.

PubMed

Voracek, Martin; Tran, Ulrich S

2007-03-01

The objective of this study was to assess the ecological association of dietary tryptophan intake and suicide rates across industrialized nations. Tryptophan, an essential amino acid, is the rate-limiting precursor of serotonin biosynthesis. The serotonergic system has been strongly implicated in the neurobiology of suicide. Contemporary male and female suicide rates for the general population (42 countries) and the elderly (38 countries) were correlated with national estimates of dietary tryptophan intake. Measures of tryptophan intake were significantly negatively associated to national suicide rates. Controlling for national affluence, total alcohol consumption and happiness levels slightly attenuated these associations, but left all of them negative. The effect is an ecological (group-level) finding. Estimated per capita tryptophan supply is only a proxy for actual consumption. Developed nations ranking high in dietary tryptophan intake rank low in suicide rates, independent of national wealth, alcohol intake and happiness.

Serotonin release varies with brain tryptophan levels

NASA Technical Reports Server (NTRS)

Schaechter, Judith D.; Wurtman, Richard J.

1990-01-01

This study examines directly the effects on serotonin release of varying brain tryptophan levels within the physiologic range. It also addresses possible interactions between tryptophan availability and the frequency of membrane depolarization in controlling serotonin release. We demonstrate that reducing tryptophan levels in rat hypothalamic slices (by superfusing them with medium supplemented with 100 microM leucine) decreases tissue serotonin levels as well as both the spontaneous and the electrically-evoked serotonin release. Conversely, elevating tissue tryptophan levels (by superfusing slices with medium supplemented with 2 microM tryptophan) increases both the tissue serotonin levels and the serotonin release. Serotonin release was found to be affected independently by the tryptophan availability and the frequency of electrical field-stimulation (1-5 Hz), since increasing both variables produced nearly additive increases in release. These observations demonstrate for the first time that both precursor-dependent elevations and reductions in brain serotonin levels produce proportionate changes in serotonin release, and that the magnitude of the tryptophan effect is unrelated to neuronal firing frequency. The data support the hypothesis that serotonin release is proportionate to intracellular serotonin levels.

Tryptophan Predicts the Risk for Future Type 2 Diabetes

PubMed Central

Chen, Tianlu; Zheng, Xiaojiao; Ma, Xiaojing; Bao, Yuqian; Ni, Yan; Hu, Cheng; Rajani, Cynthia; Huang, Fengjie; Zhao, Aihua; Jia, Weiping; Jia, Wei

2016-01-01

Recently, 5 amino acids were identified and verified as important metabolites highly associated with type 2 diabetes (T2D) development. This report aims to assess the association of tryptophan with the development of T2D and to evaluate its performance with existing amino acid markers. A total of 213 participants selected from a ten-year longitudinal Shanghai Diabetes Study (SHDS) were examined in two ways: 1) 51 subjects who developed diabetes and 162 individuals who remained metabolically healthy in 10 years; 2) the same 51 future diabetes and 23 strictly matched ones selected from the 162 healthy individuals. Baseline fasting serum tryptophan concentrations were quantitatively measured using ultra-performance liquid chromatography triple quadruple mass spectrometry. First, serum tryptophan level was found significantly higher in future T2D and was positively and independently associated with diabetes onset risk. Patients with higher tryptophan level tended to present higher degree of insulin resistance and secretion, triglyceride and blood pressure. Second, the prediction potential of tryptophan is non-inferior to the 5 existing amino acids. The predictive performance of the combined score improved after taking tryptophan into account. Our findings unveiled the potential of tryptophan as a new marker associated with diabetes risk in Chinese populations. The addition of tryptophan provided complementary value to the existing amino acid predictors. PMID:27598004

Destabilization of artificial biomembrane induced by the penetration of tryptophan

NASA Astrophysics Data System (ADS)

Chen, Liuhua; Gan, Lihua; Liu, Mingxian; Fan, Rong; Xu, Zijie; Hao, Zhixian; Chen, Longwu

2011-03-01

The effect of tryptophan on the membrane stability was studied by using three artificial biological membranes including liposome, Langmuir monolayer and solid supported bilayer lipid membrane (s-BLM) as models. All the results indicate that the penetration of tryptophan can destabilize different artificial biological membranes. The diameter of liposome and the leakage of calcein from liposome increased with the increase of tryptophan concentration because the penetration of tryptophan was beneficial for dehydrating the polar head groups of lipids and the formation of fusion intermediates. π-A isotherms of lecithin on the subphase of tryptophan solution further confirm that tryptophan can penetrate into lipid monolayer and reduce the stability of lipid monolayer. When the concentration of tryptophan increased from 0 to 2 × 10 -3 mol L -1, the limiting molecular area of lecithin increased from 110.5 to 138.5 Å 2, but the collapse pressure of the monolayer decreased from 47.6 to 42.3 mN m -1, indicating the destabilization of lipid monolayer caused by the penetration of tryptophan. The resistance spectra of s-BLM demonstrate that the existence of tryptophan leads to the formation of some defects in s-BLM and the destabilization of s-BLM. The values of electron-transfer resistance and double layer capacitance respectively decreased from 5.765 × 10 6 Ω and 3.573 × 10 -8 F to 1.391 × 10 6 Ω and 3.340 × 10 -8 F when the concentration of tryptophan increased from 0 to 2 × 10 -3 mol L -1. Correspondingly, the breakdown voltage of s-BLM decreased from 2.51 to 1.72 V.

The effect of insulin upon the influx of tryptophan into the brain of the rabbit.

PubMed

Daniel, P M; Love, E R; Moorhouse, S R; Pratt, O E

1981-03-01

1. The effect of hyperinsulinaemia upon the influx of tryptophan into the brain was determined. A raised level of insulin was maintained in the circulation of rabbits for periods of up to 120 min by means of a continuous, programmed intravenous injection of the hormone, given by an electronically controlled variable-drive syringe. A similar, appropriately programmed, intravenous injection of glucose, given simultaneously with the insulin, maintained the concentration of the blood glucose within normal limits throughout each experiment, so that the results were not vitiated by the development of hypoglycaemia. 2. Raised levels of insulin in the blood affect the supply of tryptophan to the brain in two opposing ways: (a) by increasing the binding of tryptophan to the albumin in the blood, thereby reducing the level of the free tryptophan in the circulation by about a half, which would decrease the influx of tryptophan into the brain; (b) by simultaneously reducing the levels in the blood of six or more of the amino acids which compete with tryptophan for transport carriers into the brain, which would increase the influx of tryptophan. The net result of these two opposing effects is that insulin causes only a slight increase in the influx of tryptophan into the brain. 3. To account in quantitative terms for the effect of insulin upon the influx of tryptophan into the brain it proved necessary to make one assumption. This assumption was that a predictable proportion of the tryptophan which is loosely bound to blood albumin is being stripped off this protein by the transport carrier located on the luminal surface membranes of the endothelial cells during the passage of the blood through the cerebral capillaries. If this assumption is accepted the work reported here explains adequately the effect of insulin on the influx of tryptophan into the brain.

Insight into the composition and degradation potential of dissolved organic matter with different hydrophobicity in landfill leachates.

PubMed

He, Xiao-Song; Xi, Bei-Dou; Gao, Ru-Tai; Zhang, Hui; Dang, Qiu-Ling; Li, Dan; Huang, Cai-Hong

2016-02-01

Dissolved organic matter (DOM) isolated from the leachates with different landfill ages was fractionated into hydrophobic acid (HOA), hydrophobic neutral (HON), hydrophobic base (HOB) fractions and hydrophilic matter (HIM) based on hydrophobicity, and the composition and degradation potential of the bulk DOM and its fractions were investigated by excitation-emission matrix fluorescence spectra coupled with parallel factor analysis. Results showed that the bulk DOM comprised fulvic-, humic-, tryptophan- and tyrosine-like substances, as well as component C1, whose composition and origin was unidentified. Landfill process increased the content of component C1, fulvic- and humic-like matter. The HON fractions comprised primarily component C1 and tyrosine-like matter. The HOA, HOB and HIM fractions isolated from the young leachates consisted mainly of tryptophan- and tyrosine-like substances. As to the intermediate and old leachates, the HOA and HOB fractions comprised mainly component C1, while the HIM comprised mainly fulvic-like matter. The HIM showed the most resistant against biodegradation among the four fractions, and was the main component of leachate treatment. Advanced oxidation and/or membrane treatment are recommended to remove the HIM fraction due to its hydrophilic and stable characteristics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorescence spectra of atmospheric aerosol at Adelphi, Maryland, USA: measurement and classification of single particles containing organic carbon

NASA Astrophysics Data System (ADS)

Pinnick, Ronald G.; Hill, Steven C.; Pan, Yong-Le; Chang, Richard K.

We measured laser-induced fluorescence spectra from individual supermicron-sized atmospheric particles drawn into our laboratory at Adelphi, MD, an urban site in the Washington, DC metroplex. A virtural impactor concentrator is used along with an aerodynamic-focusing-nozzle which forms, within an optical chamber, a focused aerosol jet where single aerosol particles can be interrogated on-the-fly with a pulsed 266-nm-wavelength laser. Sample rates are a few liter per minute, and are size dependent. Crossed-diode laser beams indicate when a particle is traversing the sample region and are used to trigger the UV laser to fire and the gated intensified CCD to record the fluorescence spectrum. Our breadboard fluorescence particle spectrometer measures particles in the 3-10 μm diameter size range. Typical trigger rates are a few per second. The usable spectral range is from about 295 to 605 nm. The majority of the particles have very weak fluorescence (on average 8 percent of particles have fluorescence signals above noise). The spectra were grouped using a heirarchical cluster analysis, with parameters chosen so that spectra typically cluster into 4-12 main categories. From the set of all cluster spectra we chose 8 template spectra for reanalyzing all the data. On average, 92 percent (81-94 percent) of the spectra were similar to these templates (using the same thresholds used for the cluster analysis). The major emission bands of the most commonly occurring spectra have peaks: near 460 nm (28 percent of fluorescent particles on average), a very broad hump, and may be humic acids or humic like substances; near 317 nm (on average 24 percent of fluorescent particles); near 321 and 460 nm (a double hump, 12 percent of fluorescent particles); and near 341 nm (8 percent of fluorescent particles). Some of the fluorescence in spectra peaking in the 317-341 nm range is probably from dicyclic aromatics and heterocyclics, including the amino acid tryptophan in biological particles such as bacteria and spores.

Impact of 2,4-Diacetylphloroglucinol-Producing Biocontrol Strain Pseudomonas fluorescens F113 on Intraspecific Diversity of Resident Culturable Fluorescent Pseudomonads Associated with the Roots of Field-Grown Sugar Beet Seedlings

PubMed Central

Moënne-Loccoz, Yvan; Tichy, Hans-Volker; O'Donnell, Anne; Simon, Reinhard; O'Gara, Fergal

2001-01-01

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and l-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant. PMID:11472913

Halobenzoquinone-mediated assembly of amino acid modified Mn-doped ZnS quantum dots for halobenzoquinones detection in drinking water.

PubMed

Jiao, Zhe; Zhang, Pengfei; Chen, Hongwei; Li, Jingwen; Zhong, Zhengquan; Fan, Hongbo; Cheng, Faliang

2018-10-05

Halobenzoquinones (HBQs) were reported as disinfection byproducts (DBPs) which had potential risk of bladder cancer. In this paper, a highly selective analytical method for HBQs was developed by HBQs-mediated assembly of amino acid modified Mn-doped ZnS/Quantum Dots (Mn: ZnS QDs). In the presence HBQs, a charge-transfer complex (CTC) was formed between aromatic rings of HBQs and the primary amino groups on the surface of the QDs. The formation of CTC led to the aggregation of QDs, as a result fluorescence decreasing occurred. The decrease was correlated with the concentration of HBQs. Then a fluorescence sensor array for discrimination of three kinds of HBQs including 2,6-Dichloro-1,4-benzoquinone (DCBQ), 2,6-Dibromo-1,4-benzoquinone (DBBQ) and 2,3,6-trichloro-1,4-benzoquinone (TCBQ) was developed. Four kinds of amino acids including cysteine, threonine, tyrosine and tryptophan were embellished on the Mn: ZnS QDs. The different extents of aggregation led to different fluorescence decreasing effect, thus distinct fluorescence patterns were created. It showed that three kinds of HBQs could be discriminated successfully by fluorescence sensor array at a range of concentrations through principal component analysis (PCA). The unknown samples were predicted by with a stepwise linear discriminant analysis (SLDA) using Mahalanobis distance as a selection criterion with accuracy of 100%. Remarkably, the practicability of the proposed sensor array was further validated by identification of three kinds of HBQs at different concentrations in real drinking water samples. Compared to LC/MS/MS, this fluorescent sensor array-based method was proved to be more convenient since the nanoparticles can be prepared flexibly according to the property of the target. Copyright © 2018 Elsevier B.V. All rights reserved.

Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence

NASA Astrophysics Data System (ADS)

Lammers, Ivonne; Lhiaubet-Vallet, Virginie; Ariese, Freek; Miranda, Miguel A.; Gooijer, Cees

2013-03-01

The interaction of the enantiomers of the non-steroidal anti-inflammatory drug naproxen (NPX) with human serum albumin (HSA) has been investigated using fluorescence and phosphorescence spectroscopy in the steady-state and time-resolved mode. The absorption, fluorescence excitation, and fluorescence emission spectra of (S)-NPX and (R)-NPX differ in shape in the presence of HSA, indicating that these enantiomers experience a different environment when bound. In solutions containing 0.2 M KI, complexation with HSA results in a strongly increased NPX fluorescence intensity and a decreased NPX phosphorescence intensity due to the inhibition of the collisional interaction with the heavy atom iodide. Fluorescence intensity curves obtained upon selective excitation of NPX show 8-fold different slopes for bound and free NPX. No significant difference in the binding constants of (3.8 ± 0.6) × 105 M-1 for (S)-NPX and (3.9 ± 0.6) × 105 M-1 for (R)-NPX was found. Furthermore, the addition of NPX quenches the phosphorescence of the single tryptophan in HSA (Trp-214) based on Dexter energy transfer. The short-range nature of this mechanism explains the upward curvature of the Stern-Volmer plot observed for HSA: At low concentrations NPX binds to HSA at a distance from Trp-214 and no quenching occurs, whereas at high NPX concentrations the phosphorescence intensity decreases due to dynamic quenching by NPX diffusing into site I from the bulk solution. The dynamic quenching observed in the Stern-Volmer plots based on the longest phosphorescence lifetime indicates an overall binding constant to HSA of about 3 × 105 M-1 for both enantiomers.

A combined binding mechanism of nonionic ethoxylated surfactants to bovine serum albumin revealed by fluorescence and circular dichroism.

PubMed

Iovescu, Alina; Băran, Adriana; Stîngă, Gabriela; Cantemir-Leontieş, Anca Ruxandra; Maxim, Monica Elisabeta; Anghel, Dan Florin

2015-12-01

The study systematically investigates aqueous mixtures of fixed bovine serum albumin (BSA) and various ethoxylated nonionic surfactants belonging to a homologous series or not. Mono-disperse tetra-(C12E4), hexa-(C12E6) and octa-ethyleneglycol mono-n-dodecyl ether (C12E8), and poly-disperse eicosa-ethyleneglycol mono-n-tetradecyl ether (C14EO20) are respectively employed. Fluorescence and circular dichroism measurements are performed at surfactant/protein molar ratios (rm)s lower and higher than one. We aim to get new insights into the binding mechanism of these species and to differentiate among the interaction abilities of these surfactants. The relative magnitude of the binding thermodynamic parameters by fluorescence, and the increase of α-helix prove that hydrogen bonding drives the interaction next to the hydrophobic attraction. C12En (n=4,6,8) develop more H bonds with the albumin than C14EO20 owing to a zigzag conformation of their short ethyleneoxide chains. Among the homologous surfactants, C12E6 has a slightly stronger interaction with BSA due to a maximal number of H bonds at a minimal hindering. Static fluorescence and dynamic fluorescence indicate an inter-conversion between the tryptophan (Trp) rotamers which happens around the surfactants critical micellar concentration. For C14EO20, the meander conformation of the polar group determines a less evident conversion of the Trp rotamers and smaller α-helix rise. Binding isotherms of the homologous surfactants and the fluorescence quenching mechanism by C12E6 are also provided. Copyright © 2015 Elsevier B.V. All rights reserved.

A single-sweep, nanosecond time resolution laser temperature-jump apparatus

NASA Astrophysics Data System (ADS)

Ballew, R. M.; Sabelko, J.; Reiner, C.; Gruebele, M.

1996-10-01

We describe a fast temperature-jump (T-jump) apparatus capable of acquiring kinetic relaxation transients via real-time fluorescence detection over a time interval from nanoseconds to milliseconds in a single sweep. The method is suitable for aqueous solutions, relying upon the direct absorption of laser light by the bulk water. This obviates the need for additives (serving as optical or conductive heaters) that may interact with the sample under investigation. The longitudinal temperature profile is made uniform by counterpropagating heating pulses. Dead time is limited to one period of the probe laser (16 ns). The apparatus response is tested with aqueous tryptophan and the diffusion-controlled dimerization of proflavine.

Concise, Stereocontrolled Synthesis of the Citrinadin B Core Architecture

PubMed Central

Guerrero, Carlos A.; Sorensen, Erik J.

2011-01-01

A concise, stereocontrolled synthesis of the citrinadin B core architecture from scalemic, readily available starting materials is disclosed. Highlights include ready access to both cyclic tryptophan tautomer and TRANS-2,6-disubstituted piperidine fragments, an efficient, stereoretentive mixed Claisen acylation for the coupling of these halves, and further diastereoselective carbonyl addition and oxidative rearrangement for assembly of the core. PMID:21894952

Fluorescence spectra and biological activity of aerosolized bacillus spores and MS2 bacteriophage exposed to ozone at different relative humidities in a rotating drum

NASA Astrophysics Data System (ADS)

Ratnesar-Shumate, Shanna; Pan, Yong-Le; Hill, Steven C.; Kinahan, Sean; Corson, Elizabeth; Eshbaugh, Jonathan; Santarpia, Joshua L.

2015-03-01

Biological aerosols (bioaerosols) released into the environment may undergo physical and chemical transformations when exposed to atmospheric constituents such as solar irradiation, reactive oxygenated species, ozone, free radicals, water vapor and pollutants. Aging experiments were performed in a rotating drum chamber subjecting bioaerosols, Bacillus thuringiensis Al Hakam (BtAH) spores and MS2 bacteriophages to ozone at 0 and 150 ppb, and relative humidities (RH) at 10%, 50%, and 80+%. Fluorescence spectra and intensities of the aerosols as a function of time in the reaction chamber were measured with a single particle fluorescence spectrometer (SPFS) and an Ultra-Violet Aerodynamic Particle Sizer® Spectrometer (UV-APS). Losses in biological activity were measured by culture and quantitative polymerase chain reaction (q-PCR) assay. For both types of aerosols the largest change in fluorescence emission was between 280 and 400 nm when excited at 263 nm followed by fluorescence emission between 380 and 700 nm when excited at 351 nm. The fluorescence for both BtAH and MS2 were observed to decrease significantly at high ozone concentration and high RH when excited at 263 nm excitation. The decreases in 263 nm excited fluorescence are indicative of hydrolysis and oxidation of tryptophan in the aerosols. Fluorescence measured with the UV-APS (355-nm excitation) increased with time for both BtAH and MS2 aerosols. A two log loss of MS2 bacteriophage infectivity was observed in the presence of ozone at ~50% and 80% RH when measured by culture and normalized for physical losses by q-PCR. Viability of BtAH spores after exposure could not be measured due to the loss of genomic material during experiments, suggesting degradation of extracelluar DNA attributable to oxidation. The results of these studies indicate that the physical and biological properties of bioaerosols change significantly after exposure to ozone and water vapor.

Fluorescence spectra and biological activity of aerosolized bacillus spores and MS2 bacteriophage exposed to ozone at different relative humidities in a rotating drum

DOE PAGES

Ratnesar-Shumate, Shanna; Pan, Yong-Le; Hill, Steven C.; ...

2015-10-14

Biological aerosols (bioaerosols) released into the environment may undergo physical and chemical transformations when exposed to atmospheric constituents such as solar irradiation, reactive oxygenated species, ozone, free radicals, water vapor and pollutants. Aging experiments were performed in a rotating drum chamber subjecting bioaerosols, Bacillus thuringiensis Al Hakam (BtAH) spores and MS2 bacteriophages to ozone at 0 and 150 ppb, and relative humidities (RH) at 10%, 50%, and 80+%. Fluorescence spectra and intensities of the aerosols as a function of time in the reaction chamber were measured with a single particle fluorescence spectrometer (SPFS) and an Ultra-Violet Aerodynamic Particle Sizer® Spectrometermore » (UV-APS). Losses in biological activity were measured by culture and quantitative polymerase chain reaction (q-PCR) assay. For both types of aerosols the largest change in fluorescence emission was between 280 and 400 nm when excited at 263 nm followed by fluorescence emission between 380 and 700 nm when excited at 351 nm. The fluorescence for both BtAH and MS2 were observed to decrease significantly at high ozone concentration and high RH when excited at 263 nm excitation. The decreases in 263 nm excited fluorescence are indicative of hydrolysis and oxidation of tryptophan in the aerosols. Fluorescence measured with the UV-APS (355-nm excitation) increased with time for both BtAH and MS2 aerosols. A two log loss of MS2 bacteriophage infectivity was observed in the presence of ozone at ~50% and 80% RH when measured by culture and normalized for physical losses by q-PCR. Viability of BtAH spores after exposure could not be measured due to the loss of genomic material during experiments, suggesting degradation of extracelluar DNA attributable to oxidation. The results of these studies indicate that the physical and biological properties of bioaerosols change significantly after exposure to ozone and water vapor.« less

Fluorescence spectra and biological activity of aerosolized bacillus spores and MS2 bacteriophage exposed to ozone at different relative humidities in a rotating drum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratnesar-Shumate, Shanna; Pan, Yong-Le; Hill, Steven C.

Biological aerosols (bioaerosols) released into the environment may undergo physical and chemical transformations when exposed to atmospheric constituents such as solar irradiation, reactive oxygenated species, ozone, free radicals, water vapor and pollutants. Aging experiments were performed in a rotating drum chamber subjecting bioaerosols, Bacillus thuringiensis Al Hakam (BtAH) spores and MS2 bacteriophages to ozone at 0 and 150 ppb, and relative humidities (RH) at 10%, 50%, and 80+%. Fluorescence spectra and intensities of the aerosols as a function of time in the reaction chamber were measured with a single particle fluorescence spectrometer (SPFS) and an Ultra-Violet Aerodynamic Particle Sizer® Spectrometermore » (UV-APS). Losses in biological activity were measured by culture and quantitative polymerase chain reaction (q-PCR) assay. For both types of aerosols the largest change in fluorescence emission was between 280 and 400 nm when excited at 263 nm followed by fluorescence emission between 380 and 700 nm when excited at 351 nm. The fluorescence for both BtAH and MS2 were observed to decrease significantly at high ozone concentration and high RH when excited at 263 nm excitation. The decreases in 263 nm excited fluorescence are indicative of hydrolysis and oxidation of tryptophan in the aerosols. Fluorescence measured with the UV-APS (355-nm excitation) increased with time for both BtAH and MS2 aerosols. A two log loss of MS2 bacteriophage infectivity was observed in the presence of ozone at ~50% and 80% RH when measured by culture and normalized for physical losses by q-PCR. Viability of BtAH spores after exposure could not be measured due to the loss of genomic material during experiments, suggesting degradation of extracelluar DNA attributable to oxidation. The results of these studies indicate that the physical and biological properties of bioaerosols change significantly after exposure to ozone and water vapor.« less

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

PubMed

Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

2017-08-01

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

A new approach to the old problem: Inner filter effect type I and II in fluorescence

NASA Astrophysics Data System (ADS)

Kasparek, Adam; Smyk, Bogdan

2018-06-01

The fluorescence technique is very popular and has been used in many fields of research. It is simple in its assumptions but not very easy to use. One of the main problems is the inner filter effect (IF) I and II which takes place in the cuvette. IF type I is permanently present, but IF type II occurs only when absorption and fluorescence spectra overlap. To avoid IF type I, absorbencies in the cuvette should be smaller than 0.05, which is however very difficult to obtain in many experiments. In this work we propose a new method to solve these problems in the case of a Cary Eclipse fluorimeter, having horizontally-oriented slits, based on old equations developed in the middle of the last century. This method can be applied for other instruments, even these with vertically-oriented beams, because we share scripts written in MATLAB and GRAMS/AI environment. Calculations in our method enable specifying beam geometry parameters in the cuvette, which is necessary to obtain the correct shape and fluorescence intensity of emission and excitation spectra. Such a specific fluorescence intensity dependence on absorbance can, in many cases, afford possibilities to determine the quantum yield (QY) using slopes of the straight-lines, which was demonstrated with the use of Tryptophan (Trp), Tyrosine (Tyr), and Rhodamine B (RhB) solutions. For example, assuming that QY = 0.14 for Tyr, the QY determined for RhB reached QY = 0.71 ± 0.05, although the measurement for Tyr and RhB was performed at a completely different spectral range.

Inhibition of hormonal and behavioral effects of stress by tryptophan in rats.

PubMed

Gul, Sumera; Saleem, Darakhshan; Haleem, Muhammad A; Haleem, Darakhshan Jabeen

2017-11-03

Stress in known to alter hormonal systems. Pharmacological doses of tryptophan, the essential amino acid precursor of serotonin, increase circulating leptin and decrease ghrelin in normal healthy adults. Because systemically injected leptin inhibits stress-induced behavioral deficits and systemically injected serotonin modulates leptin release from the adipocytes, we used tryptophan as a pharmacological tool to modulate hormonal and behavioral responses in unstressed and stressed rats. Leptin, ghrelin, serotonin, tryptophan, and behavior were studied in unstressed and stressed rats following oral administration of 0, 100, 200, and 300 mg/kg of tryptophan. Following oral administration of tryptophan at a dose of 300 mg/kg, circulating levels of serotonin and leptin increased and those of ghrelin decreased in unstressed animals. No effect occurred on 24-hours cumulative food intake and elevated plus maze performance. Exposure to 2 hours immobilization stress decreased 24 hours cumulative food intake and impaired performance in elevated plus maze monitored next day. Serum serotonin decreased, leptin increased, and no effect occurred on ghrelin. Stress effects on serotonin, leptin, food intake, and elevated plus maze performance did not occur in tryptophan-pretreated animals. Tryptophan-induced decreases of ghrelin also did not occur in stressed animals. The findings show an important role of serum serotonin, leptin, and ghrelin in responses to stress and suggest that the essential amino acid tryptophan can improve therapeutics in stress-induced hormonal and behavioral disorders.

24h withdrawal following repeated administration of caffeine attenuates brain serotonin but not tryptophan in rat brain: implications for caffeine-induced depression.

PubMed

Haleem, D J; Yasmeen, A; Haleem, M A; Zafar, A

1995-01-01

Caffeine injected at doses of 20, 40 and 80 mg/kg increased brain levels of tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat brain. In view of a possible role of 5-HT in caffeine-induced depression the effects of repeated administration of high doses of caffeine on brain 5-HT metabolism are investigated in rats. Caffeine was injected at doses of 80 mg/kg daily for five days. Control animals were injected with saline daily for five days. On the 6th day caffeine (80 mg/kg) injected to 5 day saline injected rats increased brain levels of tryptophan, 5-HT and 5-HIAA. Plasma total tryptophan levels were not affected and free tryptophan increased. Brain levels of 5-HT and 5-HIAA but not tryptophan decreased in 5 day caffeine injected rats injected with saline on the 6th day. Plasma total and free tryptophan were not altered in these rats. Caffeine-induced increases of brain tryptophan but not 5-HT and 5-HIAA were greater in 5 day caffeine than 5 day saline injected rats. The findings are discussed as repeated caffeine administration producing adaptive changes in the serotonergic neurons to decrease the conversion of tryptophan to 5-HT and this may precipitate depression particularly in conditions of caffeine withdrawal.

TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

PubMed Central

Freundlich, Martin; Lichstein, Herman C.

1962-01-01

Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

Chromatographic analysis of tryptophan metabolites

PubMed Central

Sadok, Ilona; Gamian, Andrzej

2017-01-01

The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach. PMID:28590049

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

PubMed

Erge, D; Schumann, B; Gröger, D

1984-01-01

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Kinetics and mechanism of the condensation of pyridoxal hydrochloride with L-tryptophan and D-tryptophan, and the chemical transformation of their products

NASA Astrophysics Data System (ADS)

Pishchugin, F. V.; Tuleberdiev, I. T.

2017-10-01

The kinetics and mechanism of interaction between pyridoxal and L-tryptophan, D-tryptophan, and their derivatives are studied. It is found that condensation reactions proceed via three kinetically distinguishable stages: (1) the rapid intraplanar addition of the NH2 groups of the amino acids to pyridoxal with the formation of amino alcohols; (2) the rotational isomerism of amino alcohol fragments with their subsequent dehydration and the formation of a Schiff base with a specific configuration; (3) the abstraction of α-hydrogen in the product of condensation of pyridoxal with L-tryptophan, or the abstraction of CO2 in the product of condensation of pyridoxal with D-tryptophan with the formation of quinoid structures, hydrolysis of which results in the preparation of pyridoxamine and keto acid or pyridoxal and tryptamine, respectively. Schiff bases resistant to further chemical transformations are formed in the reaction with tryptophan methyl ester.

Highly efficient phosphorescent, TADF, and fluorescent OLEDs (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kim, Jang-Joo; Kim, Kwon-Hyeon; Moon, Chang-Ki; Shin, Hyun

2016-09-01

High efficiency OLEDs based on phosphorescent, thermally activated delayed fluorescent (TADF) and fluorescent emitters will be presented. We will show that EQEs over 60% is achievable if OLEDs are fabricated using organic semiconductors with the refractive indices of 1.5 and fully horizontal emitting dipoles without any extra light extracting structure. We will also show that reverse intersystem crossing RISC rate plays an important role to reduce the efficiency roll-off in efficient TADF and fluorescent OLEDs and a couple to methods will be presented to increase the RISC rate in the devices.

Analysis of binding ability of two tetramethylpyridylporphyrins to albumin and its complex with bilirubin

NASA Astrophysics Data System (ADS)

Solomonov, Alexey V.; Shipitsyna, Maria K.; Vashurin, Arthur S.; Rumyantsev, Evgeniy V.; Timin, Alexander S.; Ivanov, Sergey P.

2016-11-01

An interaction between 5,10,15,20-tetrakis-(N-methyl-x-pyridyl)porphyrins, x = 2; 4 (TMPyPs) with bovine serum albumin (BSA) and its bilirubin (BR) complex was investigated by UV-Viz and fluorescence spectroscopy under imitated physiological conditions involving molecular docking studies. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, quenching sphere radius etc.) were determined. It was showed that the interaction between proteins and TMPyPs occurs via static quenching of protein fluorescence and has predominantly hydrophobic and electrostatic character. It was revealed that obtained complexes are relatively stable, but in the case of TMPyP4 binding with proteins occurs better than TMPyP2. Nevertheless, both TMPyPs have better binding ability with free protein compared to BRBSA at the same time. The influence of TMPyPs on the conformational changes in protein molecules was studied using synchronous fluorescence spectroscopy. It was found that there is no competition of BR with TMPyPs for binging sites on protein molecule and BR displacement does not occur. Molecular docking calculations have showed that TMPyPs can bind with albumin via tryptophan residue in the hydrophilic binding site of protein molecule but it is not one possible interaction way.

Effect of curcumin on amyloid-like aggregates generated from methionine-oxidized apolipoprotein A-I

DOE PAGES

Krishnamoorthy, Aparna; Tavoosi, Narjes; Chan, Gary K. L.; ...

2018-01-10

Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. Here, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A-I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid-like aggregates formed by methionine-oxidized (ox) apoA-I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of β-structure in ox-apoA-I amyloid-like aggregates formed in the presence of curcumin, comparedmore » to aggregates formed without curcumin. However, the kinetics of ox-apoA-I amyloid-like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox-apoA-I amyloid-like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA-I and alters the structure of ox-apoA-I amyloid-like aggregates yet does not diminish the propensity of ox-apoA-I to form aggregates.« less

Binding of puerarin to human serum albumin: a spectroscopic analysis and molecular docking.

PubMed

He, Yang; Wang, Yiwei; Tang, Lifei; Liu, Hui; Chen, Wei; Zheng, Zhongliang; Zou, Guolin

2008-03-01

Puerarin is a widely used compound in Chinese traditional medicine and exhibits many pharmacological activities. Binding of puerarin to human serum albumin (HSA) was investigated by ultraviolet absorbance, fluorescence, circular dichroism and molecular docking. Puerarin caused a static quenching of intrinsic fluorescence of HSA, the quenching data was analyzed by Stern-Volmer equation. There was one primary puerarin binding site on HSA with a binding constant of 4.12 x 10(4) M(-1) at 298 K. Thermodynamic analysis by Van Hoff equation found enthalpy change (DeltaH(0)) and entropy change (DeltaS(0)) were -28.01 kJ/mol and -5.63 J/mol K respectively, which indicated the hydrogen bond and Van der Waas interaction were the predominant forces in the binding process. Competitive experiments showed a displacement of warfarin by puerarin, which revealed that the binding site was located at the drug site I. Puerarin was about 2.22 nm far from the tryptophan according to the observed fluorescence resonance energy transfer between HSA and puerarin. Molecular docking suggested the hydrophobic residues such as tyrosine (Tyr) 150, Tyr 148, Tyr 149 and polar residues such as lysine (Lys) 199, Lys 195, arginine 257 and histidine 242 played an important role in the binding reaction.

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis.

PubMed

Schulze, Philipp; Ludwig, Martin; Belder, Detlev

2008-12-01

A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 microg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw- with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

Affinity to bovine serum albumin and anticancer activity of some new water-soluble metal Schiff base complexes

NASA Astrophysics Data System (ADS)

Asadi, Mozaffar; Asadi, Zahra; Zarei, Leila; Sadi, Somaye Barzegar; Amirghofran, Zahra

2014-12-01

Metal Schiff-base complexes show biological activity but they are usually insoluble in water so four new water-soluble metal Schiff base complexes of Na2[M(5-SO3-1,2-salben]; (5-SO3-1,2-salben denoted N,N";-bis(5-sulphosalicyliden)-1,2-diaminobenzylamine and M = Mg, Mn, Cu, Zn) were synthesized and characterized. The formation constants of the metal complexes were determined by UV-Vis absorption spectroscopy. The interaction of these complexes with bovine serum albumin (BSA) was studied by fluorescence spectroscopy. Type of quenching, binding constants, number of binding sites and binding stoichiometries were determined by fluorescence quenching method. The results showed that the mentioned complexes strongly bound to BSA. Thermodynamic parameters indicated that hydrophobic association was the major binding force and that the interaction was entropy driven and enthalpically disfavoured. The displacement experiment showed that these complexes could bind to the subdomain IIA (site I) of albumin. Furthermore the synchronous fluorescence spectra showed that the microenvironment of the tryptophan residues was not apparently changed. Based on the Förster theory of non-radiation energy transfer, the distance between the donor (Trp residues) and the acceptor metal complexes was obtained. The growth inhibitory effect of complexes toward the K562 cancer cell line was measured.

Interrogation of an autofluorescence-based method for protein fingerprinting.

PubMed

Siddaramaiah, Manjunath; Rao, Bola Sadashiva S; Joshi, Manjunath B; Datta, Anirbit; Sandya, S; Vishnumurthy, Vasudha; Chandra, Subhash; Nayak, Subramanya G; Satyamoorthy, Kapaettu; Mahato, Krishna K

2018-03-14

In the present study, we have designed a laser-induced fluorescence (LIF) based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, intrinsic fluorescence spectra of proteins were detected after separation on 1 or 2 dimensional Sodium Dodecyl Sulfate-Tris(2-carboxyethyl)phosphine (SDS-TCEP) polyacrylamide gel electrophoresis (PAGE) and the data were analyzed. The MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of protein after 1- and 2-dimensional protein separation. These provided objective parameters of intrinsic fluorescence intensity, emission peak, molecular weight and isoelectric point using a single platform. Further, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise were not identifiable by conventional staining, imaging and tagging methods. Categorization of the proteins based on the presence or absence of tyrosine or tryptophan residues and assigning the corresponding emission peaks (309-356 nm) with pseudo colors allowed the detection of proportion of proteins within the given spectrum. The present methodology doesn't use stains or tags, hence amenable to couple with mass spectroscopic measurements. This technique may have relevance in the field of proteomics that is used for innumerable applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Absorption and fluorescence characteristics of chromophoric dissolved organic matter in the Yangtze Estuary.

PubMed

Sun, Qiyuan; Wang, Chao; Wang, Peifang; Hou, Jun; Ao, Yanhui

2014-03-01

The Yangtze Estuary is heavily influenced by coast-continent geochemical processes and anthropogenic activity; thus, the source and distribution of chromophoric dissolved organic matter (CDOM) in the estuary are strongly impacted by these processes. Here, a series of samples were collected from across the Yangtze Estuary to investigate the source and spatial dynamics of CDOM and its components throughout the system. Three indices (a(355), spectral slope, and fluorescence) were then calculated and interpreted. The results indicated that the distribution of CDOM was dominated by allochthonous input, conservative mixing, and phase transfer. The contribution of biogenic CDOM to total CDOM increased with salinity, and three individual CDOM components were identified upon fluorescence excitation emission matrix spectroscopy and parallel factor analysis of the water samples: C1, corresponding to humic substance-like CDOM, C2, corresponding to tryptophan-like CDOM, and C3, corresponding to tyrosine-like CDOM. C1 primarily originated from a terrestrial source, C2 had widespread origins, none of which played a dominant role, and C3 mainly originated from allochthonous input in the medium salinity area. Unexpectedly, no marine humic-like component was found in the surface water of the Yangtze Estuary, possibly because turbidity decreased the depth of sunlight penetration, limiting production of this component.

Effect of curcumin on amyloid-like aggregates generated from methionine-oxidized apolipoprotein A-I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnamoorthy, Aparna; Tavoosi, Narjes; Chan, Gary K. L.

Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. Here, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A-I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid-like aggregates formed by methionine-oxidized (ox) apoA-I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of β-structure in ox-apoA-I amyloid-like aggregates formed in the presence of curcumin, comparedmore » to aggregates formed without curcumin. However, the kinetics of ox-apoA-I amyloid-like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox-apoA-I amyloid-like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA-I and alters the structure of ox-apoA-I amyloid-like aggregates yet does not diminish the propensity of ox-apoA-I to form aggregates.« less

Tryptophan metabolism via transamination. In vitro aminotransferase assay using dinitrophenylhydrazine method.

PubMed

Drsata, Jaroslav

2003-01-01

Transamination of tryptophan belongs to minor pathways of amino acid metabolism. The present paper describes conditions for application of dinitrophenylhydrazine method, originally prepared for alanine aminortansferase and aspartate aminotransferase assay, to the measurement of tryptophan transamination catalysed by any of the enzymes mentioned above. The method was tested using purified pig heart AST. While the free enzyme showed a characteristic absorption profile with the maxima at 360 and 430 nm, the course of transamination of tryptophan was confirmed by the measurement of UV-VIS spectral changes of the coenzyme in the active site of the enzyme in the presence of the amino acid substrate only, when tryptophan caused a shift of the peak from 360 nm to 330 nm due to a change of the pyridoxal form to the pyridoxamine form (= the first step of ping-pong transaminating reaction). A general limitation of dinitrophenylhydrazine method is the interference of hydrazones formed from the coenzyme pyridoxal-5'-phosphate and from the oxo- substrate 2-oxoglutarate, showing the absorption maxima at 492 nm and 388 nm, respectively with the hydrazones formed by the oxo- products (pyruvate and/or oxaloacetate in the case of ALT/AST, the absorption maxima at 443 nm in our measurements). In the case of tryptophan transamination, indolepyruvate as the oxo- product of a catalysed reaction forms dinitrophenylhydrazone, which has, besides a maximum at 435 nm, a distinct peak at 542 nm, convenient for the product concentration measurement. This is favourable for resolution from other (interfering) hydrazones. Suitable conditions for tryptophan transamination in tissue and enzyme preparations were found. Reaching optimal conditions for tryptophan transamination measurements in vitro is generally limited by low solubility of the amino acid in water solutions: With AST preparation, the velocity of catalysed reaction at 5-50 x 10(-3) M tryptophan concentration was of 1st order to the amino acid substrate. Km for tryptophan was found > or = 2 x 10(-1) M. Therefore the enzyme activity measurement at two different tryptophan concentrations is recommended for unknown samples. Tryptophan transamination by purified pig AST was compared with that catalysed by preparations obtained from mammalian tissues.

Tryptophan Intake in the US Adult Population Is Not Related to Liver or Kidney Function but Is Associated with Depression and Sleep Outcomes.

PubMed

Lieberman, Harris R; Agarwal, Sanjiv; Fulgoni, Victor L

2016-12-01

Tryptophan is an indispensable amino acid and is a precursor of the neurotransmitter serotonin. Tryptophan metabolites, such as serotonin and melatonin, are thought to participate in the regulation of mood and sleep and tryptophan is used to treat insomnia, sleep apnea, and depression. This study examined the intake of tryptophan and its associations with biochemical, behavioral, sleep, and health and safety outcomes in adults in a secondary analysis of a large, publicly available database of the US population. Data from the NHANES 2001-2012 (n = 29,687) were used to determine daily intakes of tryptophan and its associations with biochemical markers of health- and safety-related outcomes, self-reported depression, and sleep-related variables. Data were adjusted for demographic factors and protein intake. Linear trends were computed across deciles of intake for each outcome variable, and P-trends were determined. The usual tryptophan intake by US adults was 826 mg/d, severalfold higher than the Estimated Average Requirement for adults of 4 mg/(kg ⋅ d) (∼280 mg/d for a 70-kg adult). Most health- and safety-related biochemical markers of liver function, kidney function, and carbohydrate metabolism were not significantly (P-trend > 0.05) associated with deciles of tryptophan intake and were well within normal ranges, even for individuals in the 99th percentile of intake. Usual intake deciles of tryptophan were inversely associated with self-reported depression measured by the Patient Health Questionnaire raw score (0-27; P-trend < 0.01) and calculated level (1 = no depression, 5 = severe depression; P-trend < 0.01) and were positively associated with self-reported sleep duration (P-trend = 0.02). Tryptophan intake was not related to most markers of liver function, kidney function or carbohydrate metabolism. Levels of tryptophan intake in the US population appear to be safe as shown by the absence of abnormal laboratory findings. Tryptophan intake was inversely associated with self-reported level of depression and positively associated with sleep duration. © 2016 American Society for Nutrition.

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis

PubMed Central

Sherchand, Shardulendra P.; Ibana, Joyce A.; Zea, Arnold H.; Quayle, Alison J.; Aiyar, Ashok

2016-01-01

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation. PMID:27658027

Etching-dependent fluorescence quenching of Ag-dielectric-Au three-layered nanoshells: The effect of inner Ag nanosphere

NASA Astrophysics Data System (ADS)

Zhu, Jian; Xu, Zai-jie; Weng, Guo-jun; Zhao, Jing; Li, Jian-jun; Zhao, Jun-wu

2018-07-01

In this report, Ag-dielectric-Au three-layered nanoshells with controlled inner core size were synthesized. The fluorescence emission of the rhodamine 6G (R6G) could be quenched by the three-layered nanoshells distinctly. What's more, the fluorescence quenching efficiency could be further improved by tuning the etching of inner Ag nanosphere. The maximum fluorescence quenching efficiency is obtained when the separate layer just appears between the inner Ag core and the outer Au shell. Whereas the fluorescence quenching efficiency is weakened when no gaps take place around the inner Ag core or the separate layer is too thick and greater than 13 nm. The fluorescence quenching properties of the Ag-dielectric-Au three-layered nanoshells with different initial sizes of the Ag nanoparticles are also studied. The maximum fluorescence quenching efficiency is obtained when the three-layered nanoshells are synthesized based on the Ag nanoparticles with 60 nm, which is better than others two sizes (42 and 79 nm). Thus we believe that the size of initial Ag nanospheres also greatly affects the optimized fluorescence quenching efficiency. These results about fluorescence quenching properties of Ag-dielectric-Au three-layered nanoshells present a potential for design and fabrication of fluorescence nanosensors based on tuning the geometry of the inner core and the separate layer.

Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African Cornmeal Assessed in Healthy Young Men Using the Indicator Amino Acid Oxidation Technique.

PubMed

Rafii, Mahroukh; Elango, Rajavel; Ball, Ronald O; Pencharz, Paul B; Courtney-Martin, Glenda

2018-06-01

Maize is a staple food in many regions of the world, particularly in Africa and Latin America. However, maize protein is limiting in the indispensable amino acids lysine and tryptophan, making its protein of poor quality. The main objective of this study was to determine the protein quality of white African cornmeal by determining the metabolic availability (MA) of lysine and tryptophan. To determine the MA of lysine, 4 amounts of l-lysine (10, 13, 16, and 18 mg · kg-1 · d-1 totaling 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement of 35 mg · kg-1 · d-1, respectively) were studied in 6 healthy young men in a repeated-measures design. To determine the MA of tryptophan, 4 amounts of l-tryptophan (0.5, 1, 1.5, and 2 mg · kg-1 · d-1 totaling 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement of 4 mg · kg-1 · d-1, respectively) were studied in 7 healthy young men in a repeated-measures design. The MAs of lysine and tryptophan were estimated by comparing the indicator amino acid oxidation (IAAO) response with varying intakes of lysine and tryptophan in cooked white cornmeal compared with the IAAO response to l-lysine and l-tryptophan intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) with the use of the slope ratio method. The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively. Our study provides a robust estimate of the availability of lysine and tryptophan in African white maize to healthy young men. This estimate provides a basis for postproduction fortification or supplementation of maize-based diets. This trial was registered at www.clinicaltrials.gov as NCT02402179.

The Effects of Tryptophan on Everyday Interpersonal Encounters and Social Cognitions in Individuals with a Family History of Depression.

PubMed

Hogenelst, Koen; Schoevers, Robert A; Aan Het Rot, Marije

2015-03-02

Individuals with a family history of depression show subtle abnormalities in the processing of social stimuli. This could negatively affect their interpersonal functioning and contribute to their depression risk. Repeated administration of the serotonin precursor tryptophan has previously been shown to increase agreeable behavior and reduce quarrelsome behavior in irritable people, who are also considered at risk for depression. To examine the effects of tryptophan on social functioning in individuals with a family history of depression, 40 men and women with at least one first-degree relative with depression received tryptophan (1g three times a day) and placebo for 14 days each in a double-blind crossover design and recorded their social behavior and mood during everyday interpersonal encounters. Participants also provided daily ratings of their positive and negative cognitions concerning their social functioning. Tryptophan improved mood. Unexpectedly, tryptophan increased quarrelsome behavior and reduced agreeable behavior, specifically during interactions at home. The behavioral effects of tryptophan were not moderated by mood or by the interaction partner. Negative social cognitions were lower when tryptophan was given second and lower during placebo when placebo was given second. Overall, tryptophan may not alter social behavior in individuals with a family history of depression as it does in irritable people. However, the behavioral effects of tryptophan at home might be seen as a way for individuals with a family history of depression to achieve more control. Over time, this may positively influence the way they feel and think about themselves in a social context. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Relation of plasma tryptophan concentrations during pregnancy to maternal sleep and mental well-being: The GUSTO cohort.

PubMed

van Lee, Linde; Cai, Shirong; Loy, See Ling; Tham, Elaine K H; Yap, Fabian K P; Godfrey, Keith M; Gluckman, Peter D; Shek, Lynette P C; Teoh, Oon Hoe; Goh, Daniel Y T; Tan, Kok Hian; Chong, Yap Seng; Meaney, Michael J; Chen, Helen; Broekman, Birit F P; Chong, Mary F F

2018-01-01

Evidence suggests a relation between plasma tryptophan concentrations and sleep and mental well-being. As no studies have been performed in pregnant women, we studied the relation of plasma tryptophan concentrations during pregnancy with sleep quality, and mood during and after pregnancy. Pregnant women (n = 572) from the Growing Up in Singapore Towards healthy Outcomes study completed the Pittsburgh Sleep Quality Index (PSQI), the Edinburgh Postnatal Depression Scale (EPDS) and the State-Trait Anxiety Inventory (STAI) at 26-28 weeks gestation and three months post-delivery. Plasma tryptophan concentrations were measured at 26-28 weeks gestation. Poisson regressions estimated prevalence ratios (PR) for the association between tryptophan and poor sleep quality (PSQI global score > 5), probable antenatal depression (EPDS ≥ 15) and probable anxiety (STAI-state ≥ 41) were calculated adjusting for covariates. Mean plasma tryptophan concentrations was 48.0µmol/L (SD: 8.09). Higher plasma tryptophan concentrations were associated with a lower prevalence of antenatal poor sleep quality adjusting for covariates [PR: 0.88 (95% CI 0.80, 0.97) per 10µmol/L], especially in those participants who also suffered from anxiety symptoms [PR: 0.80 (95% CI 0.67, 0.95)]. No associations were observed between tryptophan concentrations during pregnancy and postnatal sleep quality or mental well-being. Subjective measures were used to assess sleep and mental well-being. We observed that higher plasma tryptophan concentrations were associated with a 12% lower prevalence of poor sleep quality during pregnancy, in particular among those with anxiety symptoms. These findings suggest the importance of having adequate tryptophan concentrations during pregnancy. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation-induced conformational changes in short peptides probed by fluorescence resonance energy transfer in the 10A domain.

PubMed

Sahoo, Harekrushna; Nau, Werner M

2007-03-26

Phosphorylation-induced conformational changes in short polypeptides were probed by a fluorescence resonance energy transfer (FRET) method by employing a short-distance FRET pair (R(0) approximately 10 A) based on tryptophan as natural donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as synthetic acceptor. Two substrates for kinases, LeuArgArgTrpSerLeuGly-Dbo (peptide I) and TrpLysArgThrLeuArgArg-Dbo (peptide II), were investigated, with serine and threonine, respectively, as phosphorylation sites. Steady-state and time-resolved fluorescence experiments in H(2)O revealed a decrease in FRET efficiency for peptide I and an increase for peptide II; this suggested that the effective distances between donor and acceptor increased and decreased, respectively. The same trends and similar absolute variations in effective donor-acceptor distances were observed in propylene glycol, a less polar and highly viscous solvent; this suggested that the variations are due to intrinsic structural preferences. Fitting of the time-resolved decay traces according to a distribution function model (Gaussian distribution) provided the mean donor-acceptor distances, which showed an increase upon phosphorylation for peptide I (from 9.7 to 10.5 A) and a decrease for peptide II (from 10.9 to 9.3 A) in H(2)O. The broadness (half-width) of the distributions, which provides a measure of the rigidity of the peptides, remained similar upon phosphorylation of peptide I (3.0 versus 3.1 A), but decreased for peptide II (from 3.1 to 0.73 A in H(2)O); this suggests a more compact, structured conformation upon phosphorylation of the latter peptide. The elongation of the peptide backbone (by ca. 0.7 A) for peptide I is attributed to an increase in steric demand upon phosphorylation, which favors an extended conformation. The contraction (by ca. 1.4 A) and structural rigidification of peptide II is attributed to attractive Coulombic interactions and hydrogen bonding between the phosphate group and the arginine residues.

Dietary tryptophan alleviates dextran sodium sulfate-induced colitis through aryl hydrocarbon receptor in mice.

PubMed

Islam, Jahidul; Sato, Shoko; Watanabe, Kouichi; Watanabe, Takaya; Ardiansyah; Hirahara, Keisuke; Aoyama, Yukihide; Tomita, Shuhei; Aso, Hisashi; Komai, Michio; Shirakawa, Hitoshi

2017-04-01

Ulcerative colitis is the typical progression of chronic inflammatory bowel disease. Amino acids, particularly tryptophan, have been reported to exert a protective effect against colitis induced by dextran sodium sulfate (DSS), but the precise underlying mechanisms remain incompletely clarified. Tryptophan metabolites are recognized to function as endogenous ligands for aryl hydrocarbon receptor (Ahr), which is a critical regulator of inflammation and immunity. Thus, we conducted this study to investigate whether dietary tryptophan supplementation protects against DSS-induced colitis by acting through Ahr. Female wild-type (WT) and Ahr-deficient (knockout; KO) mice (10-12 weeks old) were divided into four groups and fed either a control or 0.5% tryptophan diet. The tryptophan diet ameliorated DSS-induced colitis symptoms and severity in WT mice but not in KO mice, and the diet reduced the mRNA expression of Il-6, Tnfα, Il-1β and the chemokines Ccl2, Cxcl1 and Cxcl2 in the WT groups. Furthermore, Il-22 and Stat3 mRNA expression in the colon was elevated in WT mice fed with the tryptophan diet, which mainly protected epithelial layer integrity, and Ahr also modulated immune homeostasis by regulating Foxp3 and Il-17 mRNA expression. These data suggest that tryptophan-containing diet might ameliorate DSS-induced acute colitis and regulate epithelial homeostasis through Ahr. Thus, tryptophan could serve as a promising preventive agent in the treatment of ulcerative colitis. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct Secondary Structures of the Leucine-Rich Repeat Proteoglycans Decorin and Biglycan: Glycosylation-Dependent Conformational Stability

NASA Technical Reports Server (NTRS)

Krishnan, Priya; Hocking, Anne M.; Scholtz, J. Martin; Pace, C. Nick; Holik, Kimberly K.; McQuillan, David J.

1998-01-01

Biglycan and decorin, closely related small leucine-rich repeat proteoglycans, have been overexpressed in eukaryotic cers and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans. A comparative biophysical study of these glycoforms has revealed that the overall secondary structures of biglycan and decorin are different. Far-UV Circular Dichroism (CD) spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly Beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to. the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the fmal form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that at least in this specific domain, the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provided further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1-2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1-6 M urea, probably indicating the presence of stable unfolding intermediates.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

Tryptophan levels, excessive exercise, and nutritional status in anorexia nervosa.

PubMed

Favaro, A; Caregaro, L; Burlina, A B; Santonastaso, P

2000-01-01

It has been hypothesized that reduced dietary availability of tryptophan may be the cause of impaired serotonin activity in underweight anorexics. The study reported here evaluated the relationship between tryptophan availability in the blood and nutritional status in anorexia nervosa. The total amount of tryptophan and the ratio between tryptophan and other large neutral amino acids (TRP/LNAA) were assessed in a sample of 16 starving anorexic patients. Body weight and composition and energy intake were evaluated in all patients. All subjects also completed self-reported questionnaires such as the Hopkins Symptom Checklist and Eating Disorders Inventory (EDI). The TRP/LNAA ratio seems to be higher in patients with a more severe catabolic status. It is, in fact, significantly inversely correlated with body mass index, body fat, muscle mass, daily energy intake, and daily tryptophan intake. The TRP/LNAA ratio also correlates with growth hormone and the EDI drive for thinness. Patients who exercise excessively had significantly higher TRP/LNAA ratios. In starving anorexic patients, the TRP/LNAA ratio does not seem to be determined by the content of tryptophan in the diet, but it correlates with measures of catabolism. The relationship of the TRP/LNAA ratio to excessive exercise and starvation indicates the importance of further investigations exploring the role of tryptophan availability in maintaining anorexia nervosa.

Phosphorescence/microwave double-resonance spectra of tryptophan perturbed by methylmercury(II).

PubMed Central

Davis, J M; Maki, A H

1982-01-01

Amplitude-modulated phosphorescence/microwave double-resonance (AM-PMDR) spectra are reported for complexes of methylmercury(II) cation, designated CH3Hg(II), with tryptophan and glyceraldehyde-3-phosphate dehydrogenase (GPDHase; from rabbit muscle). Wavelength shifts are observed in the AM-PMDR spectra of CH3Hg(II)-tryptophan, which are obtained by microwave pumping in distinct zero-field D + E magnetic resonance transitions, demonstrating that AM-PMDR can be used to display selectively the phosphorescence spectra of structurally distinct complexes with different zero-field splittings. The AM-PMDR spectra accurately represent the phosphorescence of CH3Hg(II)-tryptophan. Binding of CH3Hg(II) to a cysteine site of GDPHase perturbs the luminescence of one of the two optically resolved tryptophan. The AM-PMDR spectrum of the perturbed tryptophan is obtained by microwave pumping of the D + E magnetic resonance signal, which can be observed optically only in the presence of a heavy atom perturbation. The resulting spectrum is broadened and shifted to the blue relative to the corresponding tryptophan phosphorescence spectrum of the uncomplexed enzyme. Comparison of the AM-PMDR spectra of CH3Hg(II)-tryptophan and CH3Hg(II)-GPDHase suggests that there are differences in the mechanisms of heavy atom perturbation in these complexes. PMID:6956860

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

NASA Technical Reports Server (NTRS)

Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

[The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase].

PubMed

Lomtev, A S; Bobrov, A G; Vekshin, N L

2004-01-01

The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Measuring peptide translocation into large unilamellar vesicles.

PubMed

Spinella, Sara A; Nelson, Rachel B; Elmore, Donald E

2012-01-27

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

The role of adrenal hormones in the activation of tryptophan 2,3-dioxygenase by nicotinic acid in rat liver.

PubMed

Sainio, E L

1997-09-01

In this study, our previous finding that nicotinic acid activates tryptophan 2,3-dioxygenase as strongly as tryptophan was investigated in further detail. This study focused on the role of the adrenals in the activation process. Adrenalectomy abolished the activation due to nicotinic acid, but not the activation caused by tryptophan. The role of corticoids and/or adrenomedullary hormones in the enzyme activation was studied, by supplementing these hormones in adrenalectomized rats using minipumps implanted under the skin. The results showed that the enhanced activity of tryptophan 2,3-dioxygenase caused by nicotinic acid was partly restored by adrenaline following adrenalectomy but not by corticosterone supplementation. The results were supported by further experiments in which the rats were treated with adrenaline or corticosterone intraperitoneally before nicotinic acid administration. The conclusion that adrenaline participates in the regulation of tryptophan 2,3-dioxygenase should promote further study to determine whether adrenaline is a general modulator of this enzyme. This experimental model generated new information on the activation mechanism of tryptophan 2,3-dioxygenase by nicotinic acid.

Degradation of microbial fluorescence biosignatures by solar ultraviolet radiation on Mars

NASA Astrophysics Data System (ADS)

Dartnell, Lewis R.; Patel, Manish R.

2014-04-01

Recent and proposed robotic missions to Mars are equipped with implements to expose or excavate fresh material from beneath the immediate surface. Once brought into the open, any organic molecules or potential biosignatures of present or past life will be exposed to the unfiltered solar ultraviolet (UV) radiation and face photolytic degradation over short time courses. The key question, then, is what is the window of opportunity for detection of recently exposed samples during robotic operations? Detection of autofluorescence has been proposed as a simple method for surveying or triaging samples for organic molecules. Using a Mars simulation chamber we conduct UV exposures on thin frozen layers of two model microorganisms, the radiation-resistant polyextremophile Deinococcus radiodurans and the cyanobacterium Synechocystis sp. PCC 6803. Excitation-emission matrices (EEMs) are generated of the full fluorescence response to quantify the change in signal of different cellular fluorophores over Martian equivalent time. Fluorescence of Deinococcus cells, protected by a high concentration of carotenoid pigments, was found to be relatively stable over 32 h of Martian UV irradiation, with around 90% of the initial signal remaining. By comparison, fluorescence from protein-bound tryptophan in Synechocystis is much more sensitive to UV photodegradation, declining to 50% after 64 h exposure. The signal most readily degraded by UV irradiation is fluorescence of the photosynthetic pigments - diminished to only 35% after 64 h. This sensitivity may be expected as the biological function of chlorophyll and phycocyanin is to optimize the harvesting of light energy and so they are readily photobleached. A significant increase in a ~450 nm emission feature is interpreted as accumulation of fluorescent cellular degradation products from photolysis. Accounting for diurnal variation in Martian sunlight, this study calculates that frozen cellular biosignatures would remain detectable by fluorescence for at least several sols; offering a sufficient window for robotic exploration operations.

Targeting Tryptophan Catabolism: A Novel Method to Block Triple-Negative Breast Cancer Metastasis

DTIC Science & Technology

2017-04-01

AWARD NUMBER: W81XWH-15-1-0039 TITLE: Targeting Tryptophan Catabolism: A Novel Method to Block Triple- Negative Breast Cancer Metastasis...Mar 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Tryptophan Catabolism: A Novel Method to Block Triple-Negative Breast Cancer...Tryptophan Catabolism: A Novel Method to Block Triple-Negative Breast Cancer Metastasis,” Submitted by Jennifer K. Richer, PhD, University of Colorado

L-Tryptophan: Effects on Daytime Sleep Latency and the Waking EEG

DTIC Science & Technology

1982-10-22

TRYPTOPHAN: EFFECTS ON DAYTIME SLEEP LATENCY AND THE WAKING EEG pr Cheryl L. Slinweber, Reidun Ursin, 1 Raymond P. Hilbert and Richard L. Hilderbrand 2 p...Gessa, 1973; Curzon & Knott , 1974; Gessa & Tagliamonte, 1974), and it has been previously suggested that 1-tryptophan may have hyp- notic effects...Curzon, G. & Knott , P.J. Fatty acids in the disposition of tryptophan. In: Aromatic Amino Acids in the Brain, Ciba Foundation Symposium 22, Elsevier

Effect of dietary supplementation of l-tryptophan on thermal tolerance and oxygen consumption rate in Cirrhinus mrigala fingerlings under varied stocking density.

PubMed

Tejpal, C S; Sumitha, E B; Pal, A K; Shivananda Murthy, H; Sahu, N P; Siddaiah, G M

2014-04-01

A 60 day feeding trial was conducted to study the effect of dietary l-tryptophan on thermal tolerance and oxygen consumption rate of freshwater fish, mrigala, Cirrhinus mrigala reared under ambient temperature at low and high stocking density. Four hundred eighty fingerlings were distributed into eight experimental groups. Four groups each of low density group (10 fishes/75L water) and higher density group (30 fishes/75L water) were fed a diet containing 0, 0.68, 1.36 or 2.72% l-tryptophan in the diet, thus forming eight experimental groups namely, Low density control (LC) (basal feed +0% l-tryptophan); LT1 (basal feed+0.68% l-tryptophan); LT2 (basal feed+1.36% l-tryptophan); LT3 (basal feed+2.72% l-tryptophan); high density control (HC) (basal feed+0% l-tryptophan); HT1 (basal feed+0.68% l-tryptophan); HT2 (basal feed+1.36% l-tryptophan); and HT3 (basal feed+2.72% l-tryptophan) were fed at 3% of the body weight. The test diets having crude protein 34.33±0.23 to 35.81±0.18% and lipid 423.49±1.76 to 425.85±0.31KCal/100g were prepared using purified ingredients. The possible role of dietary l-tryptophan on thermal tolerance and oxygen consumption rate was assessed in terms of critical thermal maxima (CTMax), critical thermal minima (CTMin), lethal thermal maxima (LTMax) and lethal thermal minima (LTMin). The CTMax, CTMin, LTMax and LTMin values were found to be significantly higher (p<0.05) in the treatment groups with CTMax 42.94±0.037 (LT2); LT Max 43.18±0.070 (LT2); CTMin 10.47±0.088 (LT2) and LTMin 9.42±0.062 (LT3), whereas the control group showed a lower tolerance level. The same trend was observed in the high density group (CTMax 42.09±0.066 (LT3); LTMax 43 23±0.067 (HT3); CTMin 10.98±0.040 (HT3) and LTMin 9.74±0.037 (HT3). However, gradual supplementation of dietary l-tryptophan in the diet significantly reduced the oxygen consumption rate in both the low density group (Y=-26.74x+222.4, r²=0.915) and the high density group (Y=-32.96x+296.5, r²=0.8923). Dietary supplementation of l-tryptophan at a level of 1.36% improved the thermal tolerance level and reduced the oxygen consumption rate in C. mrigala fingerlings. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of CDOM sources and their links with water quality in the lakes of Northeast China using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Ying; Song, Kaishan; Wen, Zhidan; Fang, Chong; Shang, Yingxin; Lv, Lili

2017-07-01

The spatial distributions of the fluorescence intensities Fmax for chromophoric dissolved organic matter (CDOM) components, the fluorescence indices (FI370 and FI310) and their correlations with water quality of 19 lakes in the Songhua River Basin (SHRB) across semiarid regions of Northeast China were examined with the data collected in September 2012 and 2015. The 19 lakes were divided into two groups according to EC (threshold value = 800 μS cm-1): fresh water (N = 13) and brackish water lakes (N = 6). The fluorescent characteristics of CDOM in the 19 lakes were investigated using excitation-emission matrix fluorescence spectroscopy (EEM) coupled with parallel factor (PARAFAC) and multivariate analysis. Two humic-like components (C1 and C3), one tryptophan-like component (C2), and one tyrosine-like component (C4) were identified by PARAFAC. The component C4 was not included in subsequent analyses due to the strong scatter in some colloidal water samples from brackish water lakes. The correlations between Fmax for the three EEM-PARAFAC extracted CDOM components C1-C3, the fluorescence indices (FI370 and FI310) and the water quality parameters (i.e., TN, TP, Chl-a, pH, EC, turbidity (Turb) and dissolved organic carbon (DOC)) were determined by redundancy analysis (RDA). The results of RDA analysis showed that spatial variation in land cover, pollution sources, and salinity/EC gradients in water quality affected Fmax for the fluorescent components C1-C3 and the fluorescence indices (FI370 and FI310). Further examination indicated that the CDOM fluorescent components and the fluorescence indices (FI370 and FI310) did not significantly differ (t-test, p > 0.05) in fresh water (N = 13) and brackish water lakes (N = 6). There was a difference in the distribution of the average Fmax for the CDOM fluorescent components between C1 to C3 from agricultural sources and urban wastewater sources in hypereutrophic brackish water lakes. The Fmax for humic-like components C1 and C3 spatially varied with land cover among the 19 lakes. Our results indicated that the spatial distributions of Fmax for CDOM fluorescent components and their correlations with water quality can be evaluated by EEM-PARAFAC and multivariate analysis among the 19 lakes across semiarid regions of Northeast China, which has potential implication for lakes with similar genesis.

[Acute tryptophan depletion in eating disorders].

PubMed

Díaz-Marsa, M; Lozano, C; Herranz, A S; Asensio-Vegas, M J; Martín, O; Revert, L; Saiz-Ruiz, J; Carrasco, J L

2006-01-01

This work describes the rational bases justifying the use of acute tryptophan depletion technique in eating disorders (ED) and the methods and design used in our studies. Tryptophan depletion technique has been described and used in previous studies safely and makes it possible to evaluate the brain serotonin activity. Therefore it is used in the investigation of hypotheses on serotonergic deficiency in eating disorders. Furthermore, and given the relationship of the dysfunctions of serotonin activity with impulsive symptoms, the technique may be useful in biological differentiation of different subtypes, that is restrictive and bulimic, of ED. 57 female patients with DSM-IV eating disorders and 20 female controls were investigated with the tryptophan depletion test. A tryptophan-free amino acid solution was administered orally after a two-day low tryptophan diet to patients and controls. Free plasma tryptophan was measured at two and five hours following administration of the drink. Eating and emotional responses were measured with specific scales for five hours following the depletion. A study of the basic characteristics of the personality and impulsivity traits was also done. Relationship of the response to the test with the different clinical subtypes and with the temperamental and impulsive characteristics of the patients was studied. The test was effective in considerably reducing plasma tryptophan in five hours from baseline levels (76%) in the global sample. The test was well tolerated and no severe adverse effects were reported. Two patients withdrew from the test due to gastric intolerance. The tryptophan depletion test could be of value to study involvement of serotonin deficits in the symptomatology and pathophysiology of eating disorders.

Tryptophan depletion disinhibits punishment but not reward prediction: implications for resilience.

PubMed

Robinson, Oliver J; Cools, Roshan; Sahakian, Barbara J

2012-01-01

We have previously shown that tryptophan depletion enhances punishment but not reward prediction (Cools et al. in Neuropsychopharmacology 33:2291-2299, 2008b). This provided evidence for a valence-specific role of serotonin (which declines under depleted tryptophan) in aversive processing. Recent theoretical (Dayan and Huys in PLoS Comput Biol 4:e4, 2008) and experimental (Crockett et al. in J Neurosci 29:11993-11999, 2009) approaches have, however, further specified this role by showing that serotonin is critical for punishment-induced inhibition. We sought to examine the role of serotonin in punishment-induced inhibition. We also examined the impact of induced mood on this effect to assess whether effects of tryptophan depletion on affective inhibition are moderated by mood. Healthy females consumed a balanced amino acid mixture with (N = 20) or without (N = 21) the serotonin precursor tryptophan. Each subject completed either negative or neutral mood induction. All subjects completed the reward and punishment reversal learning task adopted in the previous study. We demonstrate a punishment prediction impairment in individuals who consumed tryptophan which was absent in individuals who were depleted of tryptophan. This effect was impervious to mood state. Our results suggest that serotonin promotes the inhibition of responses to punishing outcomes. This may lead to reduced punishment prediction accuracy in the presence of tryptophan and may contribute to resilience to affective disorders. Reduction of serotonin via tryptophan depletion then removes this inhibition. As such, we highlight a mechanism by which reduced serotonin can contribute to disorders of impulsivity and compulsivity as well as disorders of emotion.

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Freezing-Induced Perturbation of Tertiary Structure of a Monoclonal Antibody

PubMed Central

LIU, LU; BRAUN, LATOYA JONES; WANG, WEI; RANDOLPH, THEODORE W.; CARPENTER, JOHN F.

2014-01-01

We studied the effects of pH and solution additives on freezing-induced perturbations in the tertiary structure of a monoclonal antibody (mAb) by intrinsic tryptophan fluorescence spectroscopy. In general, freezing caused perturbations in the tertiary structure of the mAb, which were reversible or irreversible depending on the pH or excipients present in the formulation. Protein aggregation occurred in freeze–thawed samples in which perturbations of the tertiary structure were observed, but the levels of protein aggregates formed were not proportional to the degree of structural perturbation. Protein aggregation also occurred in freeze–thawed samples without obvious structural perturbations, most likely because of freeze concentration of protein and salts, and thus reduced protein colloidal stability. Therefore, freezing-induced protein aggregation may or may not first involve the perturbation of its native structure, followed by the assembly processes to form aggregates. Depending on the solution conditions, either step can be rate limiting. Finally, this study demonstrates the potential of fluorescence spectroscopy as a valuable tool for screening therapeutic protein formulations subjected to freeze–thaw stress. PMID:24832730

Attomole-level protein fingerprinting based on intrinsic peptide fluorescence.

PubMed

Okerberg, E; Shear, J B

2001-04-01

Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable protein sample. We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1.5-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE). Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel. Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively. In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting. Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence. The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay.

[Linking optical properties of dissolved organic matter with NDMA formation potential in the Huangpu River].

PubMed

Dong, Qian-Qian; Zhang, Ai; Li, Yong-Mei; Chen, Ling; Huang, Qing-Hui

2014-03-01

Surface water samples from the Huangpu River were filtered to measure the UV absorption and fluorescence spectrum. Dissolved organic carbon (DOC), N-nitrosodimethylamine (NDMA), and its formation potential (NDMA-FP) were also analyzed to explore relationships between the properties of dissolved organic matter (DOM) and the formation potential of disinfection byproducts-NDMA in the Huangpu River. The study found that: NDMA-FP concentration increased with the increasing of DOC concentration (r = 0.487, P < 0.01), but it had negative relationships with SUVA254 and HIX (r = -0.605, P < 0.01; r = -0.396, P < 0.01). NDMA-FP concentration had positive relationships with the fluorescence intensity of protein-like substances such as low-molecular-weight (LMW) tyrosine-like and tryptophan-like substances (r = 0.421, P < 0.01; r = 0.426, P < 0.01), but had a negative relationship with humic-like substance (r = -0.422, P < 0.01). Therefore, NDMA formation potential increases with the increasing DOM content in the Huangpu River, which is significantly related with the protein-like substances, but decreases with the increasing aromaticity and humification of DOM.