Influence of sulfhydryl sites on metal binding by bacteria

NASA Astrophysics Data System (ADS)

Nell, Ryan M.; Fein, Jeremy B.

2017-02-01

The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5-10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified. In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0 μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low metal loading conditions, and another more abundant site that we term non-sulfhydryl sites that becomes important at high metal loadings. The resulting calculated stability constants do not vary significantly as a function of metal loading and yield reasonable fits to the observed adsorption behaviors as a function of both pH and metal loading. We use the results to calculate the speciation of metals bound by the bacterial envelope in realistic bacteria-bearing, heavy metal contaminated systems in order to demonstrate the potential importance of metal-sulfhydryl binding in the budget of bacterially-adsorbed metals under low metal-loading conditions.

Investigations on the Interactions of 5-Fluorouracil with Herring Sperm DNA: Steady State/Time Resolved and Molecular Modeling Studies

NASA Astrophysics Data System (ADS)

Chinnathambi, Shanmugavel; Karthikeyan, Subramani; Velmurugan, Devadasan; Hanagata, Nobutaka; Aruna, Prakasarao; Ganesan, Singaravelu

2015-04-01

In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103 M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluorouracil and DNA were evidenced to be groove binding and in partial intercalative mode.

Influences of urea, pH and metal ions on the interaction between cepharanthine and lysozyme by steady state fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Yumin; Li, Daojin; Xu, Chen

2015-03-01

The study on the binding mode of drug with protein is important to understand the pharmacokinetics and toxicity of the drug as well as the relationship of structure and function of the protein. In the study, the interaction between cepharanthine and lysozyme (Lys) in aqueous solution was first investigated by fluorescence spectroscopic techniques at pH 7.4. The obtained quenching rate constant and binding constant indicated the static quenching mechanism and medium binding force. The effect of cepharanthine on the conformation of Lys was analyzed using synchronous fluorescence and three-dimensional (3D) fluorescence. In addition, the effect of urea on the interaction of cepharanthine with Lys was studied and the binding capacity of cepharanthine to the denatured Lys deceases dramatically, as compared with that of cepharanthine to native Lys. Moreover, influence of pH on the interaction of cepharanthine with Lys was investigated. As compared with that at pH 7.4, the binding abilities of the drug to Lys under other pH conditions (pH 9.0, 5.5, 3.5, and 1.9) deceased. Furthermore, the effect of metal ions on the binding constant of cepharanthine with Lys was investigated.

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

Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase.

PubMed

Hiller, R; Carmeli, C

1990-07-03

The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.

The structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

PubMed Central

Middendorf, Thomas R.

2017-01-01

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components. PMID:27993951

Calorimetric and spectroscopic studies of the interaction between zidovudine and human serum albumin

NASA Astrophysics Data System (ADS)

Pîrnău, Adrian; Mic, Mihaela; Neamţu, Silvia; Floare, Călin G.; Bogdan, Mircea

2018-02-01

A quantitative analysis of the interaction between zidovudine (AZT) and human serum albumin (HSA) was achieved using Isothermal titration calorimetry (ITC) in combination with fluorescence and 1H NMR spectroscopy. ITC directly measure the heat during a biomolecular binding event and gave us thermodynamic parameters and the characteristic association constant. By fluorescence quenching, the binding parameters of AZT-HSA interaction was determined and location to binding site I of HSA was confirmed. Via T1 NMR selective relaxation time measurements the drug-protein binding extent was evaluated as dissociation constants Kd and the involvement of azido moiety of zidovudine in molecular complex formation was put in evidence. All three methods indicated a very weak binding interaction. The association constant determined by ITC (3.58 × 102 M- 1) is supported by fluorescence quenching data (2.74 × 102 M- 1). The thermodynamic signature indicates that at least hydrophobic and electrostatic type interactions played a main role in the binding process.

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase.

PubMed

Muñoz-Muñoz, Jose Luis; Garcia-Molina, Francisco; Garcia-Ruiz, Pedro Antonio; Varon, Ramon; Tudela, Jose; Rodriguez-Lopez, Jose N; Garcia-Canovas, Francisco

2011-12-01

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme. Copyright © 2011 Elsevier B.V. All rights reserved.

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

PubMed

Wszelaka-Rylik, Małgorzata; Witkiewicz-Kucharczyk, Aleksandra; Wójcik, Jacek; Bal, Wojciech

2007-05-01

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

Electrochemical and spectroscopic studies of the interaction of proflavine with DNA.

PubMed

Aslanoglu, Mehmet

2006-03-01

The interaction of proflavine with herring sperm DNA has been investigated by cyclic voltammetry and UV-Vis spectroscopy as well as viscosity measurements. Shifts in the peak potentials in cyclic voltammetry, spectral changes in UV absorption titration, an increase in viscosity of DNA and the results of the effect of ionic strength on the binding constant strongly support the intercalation of proflavine into the DNA double helix. The binding constant for the interaction between proflavine and DNA was K = 2.32 (+/- 0.41) x 10(4) M(-1) and the binding site size was 2.07 (+/- 0.1) base pairs, estimated in voltammetric measurements. The value of the binding site size was determined to be closer to that expected for a planar intercalating agent. The standard Gibbs free-energy change is ca. -24.90 kJ/mol at 25 degrees C, indicating the spontaneity of the binding interaction. The binding constant determined by UV absorption measurements was K = 2.20 (+/- 0.48) x 10(4) M(-1), which is very close to the value determined by cyclic voltammetry assuming that the binding equilibrium is static.

The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

PubMed Central

Zheng, Xiliang; Wang, Jin

2015-01-01

We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

Spectroscopic study of binding of chlorogenic acid with the surface of ZnO nanoparticles

NASA Astrophysics Data System (ADS)

Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

2017-09-01

Understanding the interaction properties of biological materials with ZnO NPs is fundamental interest in the field of biotechnological applications as well as in the formation of optoelectronic devices. In this research, the binding of ZnO NPs and chlorogenic acid (CGA) were investigated using fluorescence quenching, UV-Vis absorption spectroscopy, Fourier transform infrared (FTIR), Raman spectroscopy, scanning electron microscopy (TEM), and dynamic light scattering (DLS) techniques. The study results indicated the fluorescence quenching between ZnO NPs and CGA rationalized in terms of static quenching mechanism or the formation of nonfluorescent CGA-ZnO. From fluorescence quenching spectral analysis the binding constant ( K a ), number of binding sites ( n), and thermodynamic properties, were determined. The quenching constants ( K sv) and binding constant ( K a ), decrease with increasing the temperature and their binding sites n are 2. The thermodynamic parameters determined using Van't Hoff equation indicated binding occurs spontaneously involving the hydrogen bond and van der Walls forces played the major role in the reaction of ZnO NPs with CGA. The Raman, SEM, DLS, and Zeta potential measurements were also indicated the differences in the structure, morphology and sizes of CGA, ZnO NPs, and their corresponding CGA-ZnO due to adsorption of CGA on the surface of ZnO NPs

Copper(II) binding by dissolved organic matter: Importance of the copper-to-dissolved organic matter ratio and implications for the Biotic Ligand Model

USGS Publications Warehouse

Craven, Alison M.; Aiken, George R.; Ryan, Joseph N.

2012-01-01

The ratio of copper to dissolved organic matter (DOM) is known to affect the strength of copper binding by DOM, but previous methods to determine the Cu2+–DOM binding strength have generally not measured binding constants over the same Cu:DOM ratios. In this study, we used a competitive ligand exchange–solid-phase extraction (CLE-SPE) method to determine conditional stability constants for Cu2+–DOM binding at pH 6.6 and 0.01 M ionic strength over a range of Cu:DOM ratios that bridge the detection windows of copper-ion-selective electrode and voltammetry measurements. As the Cu:DOM ratio increased from 0.0005 to 0.1 mg of Cu/mg of DOM, the measured conditional binding constant (cKCuDOM) decreased from 1011.5 to 105.6 M–1. A comparison of the binding constants measured by CLE-SPE with those measured by copper-ion-selective electrode and voltammetry demonstrates that the Cu:DOM ratio is an important factor controlling Cu2+–DOM binding strength even for DOM isolates of different types and different sources and for whole water samples. The results were modeled with Visual MINTEQ and compared to results from the biotic ligand model (BLM). The BLM was found to over-estimate Cu2+ at low total copper concentrations and under-estimate Cu2+ at high total copper concentrations.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

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.

Kinetics and equilibria of cyanide binding to prostaglandin H synthase.

PubMed

MacDonald, I D; Dunford, H B

1989-09-01

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

Tc-99m galactosyl-neoglycoalbumin: in vitro characterization of receptor-mediated binding

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

Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.

1984-07-01

Hepatic binding protein (HBP) is a membrane receptor that binds and transports plasma glycoproteins from hepatic blood to hepatocellular lysosomes. A characterization is made of the in vitro binding of Tc-99m galactosyl-neoglycoalbumin (Tc-NGA), a synthetic HBP ligand, to liver membrane. Structural modifications of NGA resulted in the alteration of the equilibrium constant, KA, and the forward-binding rate constant, kb. Binding was second-order; the relative amount of membrane-bound NGA depended on the initial concentrations of ligand and membrane. Membrane displacement studies, using carrier ligands in contrast to previously bound Tc-NGA or I-NGA, correlated with the binding characteristics of a native HBPmore » ligand, asialo-orosomucoid. Computer simulation was used to study the detectability of the changes in HBP concentration at different values of kb. The simulations indicated that radiopharmacokinetic sensitivity to alterations in (HBP) should be possible using a neoglycoalbumin preparation with a carbohydrate density within the range of 15 to 25 galactose units per albumin molecule.« less

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

PubMed

Eble, Johannes A

2018-02-15

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

Study of fluorescence quenching of Barley α-amylase

NASA Astrophysics Data System (ADS)

Bakkialakshmi, S.; Shanthi, B.; Bhuvanapriya, T.

2012-05-01

The fluorescence quenching of Barley α-amylase by acrylamide and succinimide has been studied in water using steady-state and time-resolved fluorescence techniques. The steady-state fluorescence quenching technique has been performed in three different pHs (i.e., 6, 7 and 8) of water. Ground state and excited state binding constants (Kg &Ke) have been calculated. From the calculated binding constants (Kg &Ke) the free energy changes for the ground (ΔGg) and excited (ΔGe) states have been calculated and are presented in tables. UV and FTIR spectra have also been recorded to prove the binding of Barley α-amylase with acrylamide and succinimide.

[Derivative spectrophotometric and NMR spectroscopic study in pharmaceutical science].

PubMed

Kitamura, Keisuke

2007-10-01

This review starts with an introduction of derivative spectrophotometry followed by a description on the construction of a personal computer-assisted derivative spectrophotometric (DS) system. An acquisition system for inputting digitalized absorption spectra into personal computers and a BASIC program for calculating derivative spectra were developed. Then, applications of the system to drug analyses that are difficult with traditional absorption methods are described. Following this, studies on the interactions of drugs with biological macromolecules by the DS and NMR methods were discussed. An (1)H NMR study elucidated that the small unilamellar vesicle (SUV) has a single membrane made of a phosphatidylcholine bilayer, and that chlorpromazine interacts with both the outer and inner layers. (13)C NMR revealed a reduction of the dissociation constants of phenothiazine drugs due to their interaction with SUV. The partition coefficients of phenothiazine, benzodiazepine and steroid drugs in an SUV-water system and the effects of cholesterol or amino lipids content on these partition coefficients were examined by the DS method. The binding constants of phenothiazine drugs to bovine serum albumin (BSA) and the influence of Na(+), K(+), Cl(-), Br(-), and I(-) on these binding constants were determined by DS. It was found that I(-), Br(-), Cl(-) reduce the binding constants in this order, and that Na(+) and K(+) have no effect. A (19)F NMR study revealed that triflupromazine binds to BSA and human serum albumin in two regions including Site II with different populations, and that a nonsteroidal anti-inflammatory drug, niflumic acid, binds Sites Ia and Ib.

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin.

PubMed

Xu, Yujing; Hong, Tingting; Chen, Xueping; Ji, Yibing

2017-05-01

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S-omeprazole, S-OME) and its R-enantiomer (R-omeprazole, R-OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10 3 M -1 and 5.36 × 10 3 M -1 , respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins

NASA Astrophysics Data System (ADS)

Ma, Hong-Min; Chen, Xin; Zhang, Nuo; Han, Yan-Yan; Wu, Dan; Du, Bin; Wei, Qin

2009-04-01

The interaction of a water-soluble cationic porphyrin, meso-tetrakis (4- N, N, N-trimethylanilinium) porphyrin (TMAP), with two proteins, bovine serum albumin (BSA) and human serum albumin (HSA), was studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, fluorescence anisotropy and synchronous fluorescence spectroscopy at neutral aqueous solutions. Free base TMAP bound to proteins as monomers and no aggregation was observed. The binding of TMAP quenched the fluorescence of the protein. On the contrary, the fluorescence of TMAP was enhanced and the fluorescence anisotropy increased due to the binding. The direct static binding mechanism could account for the quenching by TMAP and the binding constants were calculated. TMAP showed a higher quenching efficiency and binding constant of HSA than BSA. The binding of TMAP had no obvious effect on the molecular conformation of the protein. There was only one binding site for TMAP and it was located on the surface of the protein molecule. Electrostatic force played an important role in the binding due to the opposite charges on porphyrin and the proteins.

Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins.

PubMed

Ma, Hong-Min; Chen, Xin; Zhang, Nuo; Han, Yan-Yan; Wu, Dan; Du, Bin; Wei, Qin

2009-04-01

The interaction of a water-soluble cationic porphyrin, meso-tetrakis (4-N,N,N-trimethylanilinium) porphyrin (TMAP), with two proteins, bovine serum albumin (BSA) and human serum albumin (HSA), was studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, fluorescence anisotropy and synchronous fluorescence spectroscopy at neutral aqueous solutions. Free base TMAP bound to proteins as monomers and no aggregation was observed. The binding of TMAP quenched the fluorescence of the protein. On the contrary, the fluorescence of TMAP was enhanced and the fluorescence anisotropy increased due to the binding. The direct static binding mechanism could account for the quenching by TMAP and the binding constants were calculated. TMAP showed a higher quenching efficiency and binding constant of HSA than BSA. The binding of TMAP had no obvious effect on the molecular conformation of the protein. There was only one binding site for TMAP and it was located on the surface of the protein molecule. Electrostatic force played an important role in the binding due to the opposite charges on porphyrin and the proteins.

Interaction of Antiinflammatory Drugs with EPC Liposomes: Calorimetric Study in a Broad Concentration Range

PubMed Central

Matos, Carla; Lima, José L. C.; Reis, Salette; Lopes, António; Bastos, Margarida

2004-01-01

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (Kp), and/or the equilibrium constant (Kb). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, ΔH, and indirectly, ΔG and ΔS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction—possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug. PMID:14747330

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

PubMed

Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

2014-01-01

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

PubMed

Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

2013-07-01

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration.

PubMed

Kahle, Claudia; Holzgrabe, Ulrike

2004-10-01

Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

Mussel-inspired histidine-based transient network metal coordination hydrogels

PubMed Central

Fullenkamp, Dominic E.; He, Lihong; Barrett, Devin G.; Burghardt, Wesley R.; Messersmith, Phillip B.

2013-01-01

Transient network hydrogels cross-linked through histidine-divalent cation coordination bonds were studied by conventional rheologic methods using histidine-modified star poly(ethylene glycol) (PEG) polymers. These materials were inspired by the mussel, which is thought to use histidine-metal coordination bonds to impart self-healing properties in the mussel byssal thread. Hydrogel viscoelastic mechanical properties were studied as a function of metal, pH, concentration, and ionic strength. The equilibrium metal-binding constants were determined by dilute solution potentiometric titration of monofunctional histidine-modified methoxy-PEG and were found to be consistent with binding constants of small molecule analogs previously studied. pH-dependent speciation curves were then calculated using the equilibrium constants determined by potentiometric titration, providing insight into the pH dependence of histidine-metal ion coordination and guiding the design of metal coordination hydrogels. Gel relaxation dynamics were found to be uncorrelated with the equilibrium constants measured, but were correlated to the expected coordination bond dissociation rate constants. PMID:23441102

Analysis of cholera toxin-ganglioside interactions by flow cytometry.

PubMed

Lauer, Sabine; Goldstein, Byron; Nolan, Rhiannon L; Nolan, John P

2002-02-12

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.

New mixed ligand palladium(II) complexes based on the antiepileptic drug sodium valproate and bioactive nitrogen-donor ligands: Synthesis, structural characterization, binding interactions with DNA and BSA, in vitro cytotoxicity studies and DFT calculations

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; Tavakol, Hossein

2015-04-01

The complexes [Pd(valp)2(imidazole)2] (1), [Pd(valp)2(pyrazine)2] (2) (valp is sodium valproate) have been synthesized and characterized using IR, 1H NMR, 13C{1H} NMR and UV-Vis spectrometry. The interaction of complexes with CT-DNA has been investigated using spectroscopic tools and viscosity measurement. In each case, the association constant (Kb) was deduced from the absorption spectral study and the number of binding sites (n) and the binding constant (K) were calculated from relevant fluorescence quenching data. As a result, a non-covalent interaction between the metal complex and DNA was suggested, which could be assigned to an intercalative binding. In addition, the interaction of 1 and 2 was ventured with bovine serum albumin (BSA) with the help of absorption and fluorescence spectroscopy measurements. Through these techniques, the apparent association constant (Kapp) and the binding constant (K) could be calculated for each complex. Evaluation of cytotoxic activity of the complexes against four different cancer cell lines proved that the complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate. Moreover, density functional theory (DFT) calculations were employed to provide more evidence about the observed data. The majority of trans isomers were supported not only by energies, but also by the similarity of its calculated IR frequencies, UV adsorptions and NMR chemical shifts to the experimental values.

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.

Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

2011-03-01

Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

ERIC Educational Resources Information Center

Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

2006-01-01

The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Pedersen, A O; Galliano, M; Minchiotti, L; Brennan, S O; Tárnoky, A L; Franco, M H; Salzano, F M

1996-12-15

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

PROFEAT Update: A Protein Features Web Server with Added Facility to Compute Network Descriptors for Studying Omics-Derived Networks.

PubMed

Zhang, P; Tao, L; Zeng, X; Qin, C; Chen, S Y; Zhu, F; Yang, S Y; Li, Z R; Chen, W P; Chen, Y Z

2017-02-03

The studies of biological, disease, and pharmacological networks are facilitated by the systems-level investigations using computational tools. In particular, the network descriptors developed in other disciplines have found increasing applications in the study of the protein, gene regulatory, metabolic, disease, and drug-targeted networks. Facilities are provided by the public web servers for computing network descriptors, but many descriptors are not covered, including those used or useful for biological studies. We upgraded the PROFEAT web server http://bidd2.nus.edu.sg/cgi-bin/profeat2016/main.cgi for computing up to 329 network descriptors and protein-protein interaction descriptors. PROFEAT network descriptors comprehensively describe the topological and connectivity characteristics of unweighted (uniform binding constants and molecular levels), edge-weighted (varying binding constants), node-weighted (varying molecular levels), edge-node-weighted (varying binding constants and molecular levels), and directed (oriented processes) networks. The usefulness of the network descriptors is illustrated by the literature-reported studies of the biological networks derived from the genome, interactome, transcriptome, metabolome, and diseasome profiles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative evaluation of in vitro efficacy of colesevelam hydrochloride tablets.

PubMed

Krishnaiah, Yellela S R; Yang, Yongsheng; Bykadi, Srikant; Sayeed, Vilayat A; Khan, Mansoor A

2014-09-01

Colesevelam hydrochloride is used as an adjunct to diet and exercise to reduce elevated low-density lipoprotein (LDL) cholesterol in patients with primary hyperlipidemia as well as to improve glycemic control in patients with type 2 diabetes. This is likely to result in submission of abbreviated new drug applications (ANDA). This study was conducted to compare the efficacy of two tablet products of colesevelam hydrochloride based on the in vitro binding of bile acid sodium salts of glycocholic acid (GC), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA). Kinetic binding study was carried out with constant initial bile salt concentrations as a function of time. Equilibrium binding studies were conducted under conditions of constant incubation time and varying initial concentrations of bile acid sodium salts. The unbound concentration of bile salts was determined in the samples of these studies. Langmuir equation was utilized to calculate the binding constants k1 and k2. The amount of the three bile salts bound to both the products reached equilibrium at 3 h. The similarity factor (f2) was 99.5 based on the binding profile of total bile salts to the test and reference colesevelam tablets as a function of time. The 90% confidence interval for the test to reference ratio of k2 values were 96.06-112.07 which is within the acceptance criteria of 80-120%. It is concluded from the results that the test and reference tablets of colesevelam hydrochloride showed a similar in vitro binding profile and capacity to bile salts.

Spectrophotometric study on binding of 2-thioxanthone acetic acid with ct-DNA.

PubMed

Ataci, Nese; Ozcelik, Elif; Arsu, Nergis

2018-06-02

Thioxanthone and its derivatives are the most remarkable molecules due to their vast variety of application such as radiation curing that is, until using them as a therapeutic drug. Therefore, in this study it was intended to use 2-Thioxanthone acetic acid with and without NaCl in Tris HCl buffer solution (pH:7.0) to represent the interaction with ct-DNA. The UV-vis absorption spectra of TXCH 2 COOH in the presence of ct-DNA showed hypochromism and the intrinstic binding constant (K b ) was determined as 6 × 10 3  L mol -1 . The fluoresence intensity of TXCH 2 COOH with ct-DNA clearly increased up to 101% which indicated that the fluorescence intensity was very sensitive to ct-DNA concentration. The binding constant (K) and the values of number of binding sites (n) and were calculated as 1.8 × 10 3  L mol -1 and 0.69, respectively. When the quenching constants (K sv ) of free TXCH 2 COOH and TXCH 2 COOH, which were bonded with ct-DNA were compared, slightly changed values of Ksv were seen. Moreover, displacement assay with Hoechst 33,258 and viscosity measurements in the presence and absence of NaCl salt also confirmed the binding mode which noted the electrostatic interaction following groove binding between TXCH 2 COOH and ct-DNA. Last but not least, the salt effect was examined on ct-DNA binding with TXCH 2 COOH. The results of the experiments indicated that the groove binding was strengthened by NaCl whereas in the high NaCl concentration, the binding ability of TXCH 2 COOH to ct-DNA was inversely affected. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Label-Free Determination of Protein Binding in Aqueous Solution using Overlayer Enhanced Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (OE-ATR-FTIR)

NASA Astrophysics Data System (ADS)

Ruthenburg, Travis; Aweda, Tolulope; Park, Simon; Meares, Claude; Land, Donald

2009-03-01

Protein binding/affinity studies are often performed using Surface Plasmon Resonance techniques that don't produce much spectral information. Measurement of protein binding affinity using FTIR is traditionally performed using high protein concentration or deuterated solvent. By immobilizing a protein near the surface of a gold-coated germanium internal reflection element interactions can be measured between an immobilized protein and free proteins or small molecules in aqueous solution. By monitoring the on and off rates of these interactions, the dissociation constant for the system can be determined. The dissociation constant for the molecule Yttrium-DOTA binding to the antibody 2D12.5 system was determined to be 100nM. Results will also be presented from our measurements of Bovine Serum Albumin (BSA) binding to anti-BSA.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of solute-binding properties of plastic materials used as pharmaceutical product containers.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy

2010-01-01

Material/water equilibrium binding constants (E(b)) were determined for 11 organic solutes and 2 plastic materials commonly used in pharmaceutical product containers (plasticized polyvinyl chloride and polyolefin). In general, solute binding by the plasticized polyvinyl chloride material was greater, by nearly an order of magnitude, than the binding by the polyolefin (on an equal weight basis). The utilization of the binding constants to facilitate container compatibility assessments (e.g., drug loss by container binding) for drug-containing products is discussed.

Simultaneous effects of temperature and pressure on the donor binding energy in a V-groove quantum wire

NASA Astrophysics Data System (ADS)

Khordad, R.

2010-03-01

The influence of temperature and pressure, simultaneously, on the binding energy of a hydrogenic donor impurity in a ridge GaAs/Ga 1- xAl xAs quantum wire is studied using a variational procedure within the effective mass approximation. The subband energy and the binding energy of the donor impurity in its ground state as a function of the wire bend width and impurity location at different temperatures and pressures are calculated. The results show that, when the temperature increases, the donor binding energy decreases for a constant applied pressure for all wire bend widths. Also, the binding energy increases by increasing the pressure for a constant temperature for all wire bend widths. In addition, when the temperature and pressure are applied simultaneously the binding energy decreases as the quantum wire bend width increases. On the whole, it is deduced that the temperature and pressure have important effects on the donor binding energy in a V-groove quantum wire.

The Use of Hammett Constants to Understand the Non-Covalent Binding of Aromatics

PubMed Central

Lewis, Michael; Bagwill, Christina; Hardebeck, Laura K. E.; Wireduaah, Selina

2012-01-01

Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions. PMID:24688634

Mechanism of host-guest complexation by cucurbituril.

PubMed

Márquez, César; Hudgins, Robert R; Nau, Werner M

2004-05-12

The factors affecting host-guest complexation between the molecular container compound cucurbit[6]uril (CB6) and various guests in aqueous solution are studied, and a detailed complexation mechanism in the presence of cations is derived. The formation of the supramolecular complex is studied in detail for cyclohexylmethylammonium ion as guest. The kinetics and thermodynamics of complexation is monitored by NMR as a function of temperature, salt concentration, and cation size. The binding constants and the ingression rate constants decrease with increasing salt concentration and cation-binding constant, in agreement with a competitive binding of the ammonium site of the guest and the metal cation with the ureido carbonyl portals of CB6. Studies as a function of guest size indicate that the effective container volume of the CB6 cavity is approximately 105 A(3). It is suggested that larger guests are excluded for two reasons: a high activation barrier for ingression imposed by the tight CB6 portals and a destabilization of the complex due to steric repulsion inside. For example, in the case of the nearly spherical azoalkane homologues 2,3-diazabicyclo[2.2.1]hept-2-ene (DBH, volume ca. 96 A(3)) and 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO, volume ca. 110 A(3)), the former forms the CB6 complex promptly with a sizable binding constant (1300 M(-1)), while the latter does not form a complex even after several months at optimized complexation conditions. Molecular mechanics calculations are performed for several CB6/guest complexes. A qualitative agreement is found between experimental and calculated activation energies for ingression as a function of both guest size and state of protonation. The potential role of constrictive binding by CB6 is discussed.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

Binding of resveratrol to the minor groove of DNA sequences with AATT and TTAA segments induces differential stability.

PubMed

Nair, Maya S; D'Mello, Samar; Pant, Rashmi; Poluri, Krishna Mohan

2017-05-01

Interactions of a natural stilbene compound, resveratrol with two DNA sequences containing AATT/TTAA segments have been studied. Resveratrol is found to interact with both the sequences. The mode of interaction has been studied using absorption, steady state fluorescence and circular dichroism spectroscopic techniques. UV-visible absorption and fluorescence studies provided the information regarding the binding constants and the stoichiometry of binding, whereas circular dichroism studies depicted the structural changes in DNA upon resveratrol binding. Our results evidenced that, though resveratrol showed similar affinity to both the sequences, the mode of interactions was different. The binding constants of resveratrol to AATT/TTAA sequences were found to be 7.55×10 5 M -1 and 5.42×10 5 M -1 respectively. Spectroscopic data evidenced for a groove binding interaction. Melting studies showed that the binding of resveratrol induces differential stability to the DNA sequences d(CGTTAACG) 2 and d(CGAATTCG) 2 . Fluorescence data showed a stoichiometry of 1:1 for d(CGAATTCG) 2 -resveratrol complex and 1:4 for d(CGTTAACG) 2 -resveratrol complex. Molecular docking studies demonstrated that resveratrol binds to the minor groove region of both the sequences to form stable complexes with varied atomic contacts to the DNA bases or backbone. Both the complexes are stabilized by hydrogen bond formation. Our results evidenced that modulation of DNA sequence within the same bases can greatly alter the binding geometry and stability of the complex upon binding to small molecule inhibitor compounds like resveratrol. Copyright © 2017 Elsevier B.V. All rights reserved.

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

PubMed

Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

2007-05-15

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study.

PubMed

Moeinpour, Farid; Mohseni-Shahri, Fatemeh S; Malaekeh-Nikouei, Bizhan; Nassirli, Hooriyeh

2016-09-25

The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

PubMed

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Energetics and kinetics of cooperative cofilin-actin filament interactions.

PubMed

Cao, Wenxiang; Goodarzi, Jim P; De La Cruz, Enrique M

2006-08-11

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.7 thermodynamically bound counterions. Counterion dissociation contributes approximately 40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

USGS Publications Warehouse

Uhle, M.E.; Chin, Y.-P.; Aiken, G.R.; McKnight, Diane M.

1999-01-01

Two ortho- (2,2',5 and 2,2',5,6') and a non-ortho- (3,3',4,4') substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho- substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1' carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.Two ortho- (2,2???,5 and 2,2???,5,6???) and a non-ortho- (3,3???,4,4???) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1??? carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.

Evidence of bovine serum albumin-viologen herbicide binding interaction and associated structural modifications

NASA Astrophysics Data System (ADS)

Roy, Swarup; Saxena, Shailendra K.; Mishra, Suryakant; Yogi, Priyanka; Sagdeo, P. R.; Kumar, Rajesh

2017-07-01

The binding ability of viologen herbicide with bovine serum albumin (BSA) has been investigated to understand viologen associated hazards by investigating ethyl viologen's (EV) binding using various spectroscopies and in-silico molecular docking approaches. Apparent association constant (1.3 × 104 L/mol), calculated using UV-Vis spectra indicating a moderate complex formation between BSA and EV. A static mode of fluorescence quenching has been observed as evident from inverse temperature dependence of Stern-Volmer quenching constant which also confirms an EV-BSA complex formation. Emission and time resolved fluorescence studies reveal that the emission quenching of BSA with EV is initiated by static quenching mechanism. A moderately strong binding affinity between EV and BSA has been observed (binding constant value of 7.58 × 104 L/Mol) using fluorescence quenching titration, obtained at 298 K. Quantitative measurements of thermodynamic parameters like enthalpy and entropy changes clearly indicates hydrophobic force responsible for EV-BSA complex formation. The binding distance between EV and BSA was found to be 4.48 nm are involved in non-radiative energy transfer process. Furthermore, from the circular dichroism spectra it was observed that addition of EV is also found to change the secondary structure of BSA which leads to decrease in α-helix. Above mentioned results are found to be in consonance with molecular docking simulations and supports the EV-BSA binding.

Which model based on fluorescence quenching is suitable to study the interaction between trans-resveratrol and BSA?

NASA Astrophysics Data System (ADS)

Wei, Xin Lin; Xiao, Jian Bo; Wang, Yuanfeng; Bai, Yalong

2010-01-01

There are several models by means of quenching fluorescence of BSA to determine the binding parameters. The binding parameters obtained from different models are quite different from each other. Which model is suitable to study the interaction between trans-resveratrol and BSA? Herein, twelve models based fluorescence quenching of BSA were compared. The number of binding sites increasing with increased binding constant for similar compounds binding to BSA maybe one approach to resolve this question. For example, here eleven flavonoids were tested to illustrate that the double logarithm regression curve is suitable to study binding polyphenols to BSA.

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015.

PubMed

Falconer, Robert J

2016-10-01

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x-ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion-π and π-π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Modeling the Concentrations and Efficiencies for the Interacting Species of Pyropheophorbide Methyl Ester-Copper Association

NASA Astrophysics Data System (ADS)

Al-Omari, S.

2013-07-01

The interaction between pyropheophorbide methyl ester (PPME) and Cu2+ was investigated using UV-vis and fluorescence spectrscopy. Study of the binding interaction between PPME and Cu2+ could contribute to understanding of its pharmacokinetics and pharmacodynamics. Parameters of the static and dynamic fluorescence quenching of PPME-Cu2+ association were calculated at different temperatures. For binding site of 1:1 at 299 K, the static binding constant (kS), the static isosbestic concentration (CS{ iso}), the dynamic binding constant (kD), and the dynamic isosbestic concentration (CD{ iso }) are, respectively, 61 M-1, 0.0164 M, 75 M-1, and 0.0133 M. The concentrations and efficiencies of the intermediates species were modeled. Satisfactory correspondence between the experimental and calculated results was found.

Spectroscopic investigation on the mechanism of formation of molecular complexes of albendazole and trimethoprim with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

NASA Astrophysics Data System (ADS)

Ganesh, K.; Balraj, C.; Satheshkumar, A.; Elango, K. P.

2012-06-01

UV-vis, 1H NMR, FT-IR, mass and fluorescence spectral techniques were employed to investigate the mechanism of interaction of albendazole and trimethoprim with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and to characterize the reaction products. The interaction of DDQ with trimethoprim (TMP) and albenadazole (ALB) were found to proceed through the formation of donor-acceptor complex, containing DDQ radical anion and its conversion to the product. Fluorescence quenching studies indicated that the interaction between the donors and the acceptor are spontaneous and the interaction of TMP-DDQ (binding constant = 2.9 × 105) is found to be stronger than that the ALB-DDQ (binding constant = 3 × 103) system. Also, the binding constant increased with an increase in polarity of the medium indicating the involvement of radical anion as intermediate.

Correlation between cross-bridge kinetics obtained from Trp fluorescence of myofibril suspensions and mechanical studies of single muscle fibers in rabbit psoas.

PubMed

Candau, Robin; Kawai, Masataka

2011-12-01

Our goal is to correlate kinetic constants obtained from fluorescence studies of myofibril suspension with those from mechanical studies of skinned muscle fibers from rabbit psoas. In myofibril studies, the stopped-flow technique with tryptophan fluorescence was used; in muscle fiber studies, tension transients with small amplitude sinusoidal length perturbations were used. All experiments were performed using the equivalent solution conditions (200 mM ionic strength, pH 7.00) at 10°C. The concentration of MgATP was varied to characterize kinetic constants of the ATP binding step 1 (K (1): dissociation constant), the binding induced cross-bridge detachment step 2 (k (2), k (-2): rate constants), and the ATP cleavage step 3 (k (3), k (-3)). In myofibrils we found that K (1) = 0.52 ± 0.08 mM (±95% confidence limits), k (2) = 242 ± 24 s(-1), and k (-2) ≈ 0; in muscle fibers, K (1) = 0.46 ± 0.06 mM, k (2) = 286 ± 32 s(-1), and k (-2) = 57 ± 21 s(-1). From these results, we conclude that myofibrils and muscle fibers exhibit nearly equal ATP binding step, and nearly equal ATP binding induced cross-bridge detachment step. Consequently, there is a good correlation between process C (phase 2 of step analysis) and the cross-bridge detachment step. The reverse detachment step is finite in fibers, but almost absent in myofibrils. We further studied partially cross-linked myofibrils and found little change in steps 2 and 3, indicating that cross-linking does not affect these steps. However, we found that K (1) is 2.5× of native myofibrils, indicating that MgATP binding is weakened by the presence of the extra load. We further studied the phosphate (Pi) effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP, with the inhibitory dissociation constant of ~9 mM. Similar results were also deduced from fiber studies. To characterize the ATP cleavage step in myofibrils, we measured the slow rate constant in fluorescence, and found that k (3) + k (-3) = 16 ± 1 s(-1).

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

PubMed

Collauto, Alberto; DeBerg, Hannah A; Kaufmann, Royi; Zagotta, William N; Stoll, Stefan; Goldfarb, Daniella

2017-06-14

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

PubMed

Beeram, Sandya R; Zheng, Xiwei; Suh, Kyungah; Hage, David S

2018-03-03

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha 1 -acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered. Copyright © 2018 Elsevier Inc. All rights reserved.

Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

PubMed

Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

2017-10-01

Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

PubMed

Nag, Moupriya; Bera, Kallol; Chakraborty, Sandipan; Basak, Soumen

2013-10-05

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado. Copyright © 2013 Elsevier B.V. All rights reserved.

A hierarchical approach to cooperativity in macromolecular and self-assembling binding systems.

PubMed

Garcés, Josep Lluís; Acerenza, Luis; Mizraji, Eduardo; Mas, Francesc

2008-04-01

The study of complex macromolecular binding systems reveals that a high number of states and processes are involved in their mechanism of action, as has become more apparent with the sophistication of the experimental techniques used. The resulting information is often difficult to interpret because of the complexity of the scheme (large size and profuse interactions, including cooperative and self-assembling interactions) and the lack of transparency that this complexity introduces into the interpretation of the indexes traditionally used to describe the binding properties. In particular, cooperative behaviour can be attributed to very different causes, such as direct chemical modification of the binding sites, conformational changes in the whole structure of the macromolecule, aggregation processes between different subunits, etc. In this paper, we propose a novel approach for the analysis of the binding properties of complex macromolecular and self-assembling systems. To quantify the binding behaviour, we use the global association quotient defined as K(c) = [occupied sites]/([free sites] L), L being the free ligand concentration. K(c) can be easily related to other measures of cooperativity (such as the Hill number or the Scatchard plot) and to the free energies involved in the binding processes at each ligand concentration. In a previous work, it was shown that K(c) could be decomposed as an average of equilibrium constants in two ways: intrinsic constants for Adair binding systems and elementary constants for the general case. In this study, we show that these two decompositions are particular cases of a more general expression, where the average is over partial association quotients, associated with subsystems from which the system is composed. We also show that if the system is split into different subsystems according to a binding hierarchy that starts from the lower, microscopic level and ends at the higher, aggregation level, the global association quotient can be decomposed following the hierarchical levels of macromolecular organisation. In this process, the partial association quotients of one level are expressed, in a recursive way, as a function of the partial quotients of the level that is immediately below, until the microscopic level is reached. As a result, the binding properties of very complex macromolecular systems can be analysed in detail, making the mechanistic explanation of their behaviour transparent. In addition, our approach provides a model-independent interpretation of the intrinsic equilibrium constants in terms of the elementary ones.

Investigation of flavonoids bearing different substituents on ring C and their cu2+ complex binding with bovine serum albumin: structure-affinity relationship aspects.

PubMed

Shi, Shuyun; Zhang, Yuping; Chen, Xiaoqin; Peng, Mijun

2011-10-12

The effects of 1:1 flavonoid-Cu(2+) complexes of four flavonoids with different C-ring substituents, quercetin (QU), luteolin (LU), taxifolin (TA), and (+)-catechin (CA), on bovine serum albumin (BSA) were investigated and compared with corresponding free flavonoids by spectroscopic analysis in an attempt to characterize the chemical association taking place. The results indicated that all of the quenching mechanisms were based on static quenching combined with nonradiative energy transfer. Cu(2+) chelation changed the binding constants for BSA depending on the structures of flavonoids and the detected concentrations. The reduced hydroxyl groups, increased steric hindrance, and hydrophilicity of Cu(2+) chelation may be the main reasons for the reduced binding constants, whereas the formation of stable flavonoid-Cu(2+) complexes and synergistic action could increase the binding constants. The changed trends of critical energy transfer distance (R(0)) for Cu(2+) chelation were contrary to those of binding constants.

Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Brennan, S O; Minchiotti, L; Galliano, M

1994-07-01

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

NASA Astrophysics Data System (ADS)

Kabir, Md. Zahirul; Tee, Wei-Ven; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad

2017-06-01

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04 × 104 M-1), obtained at 298 K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.

Calcium ion binding to a soil fulvic acid using a donnan potential model

USGS Publications Warehouse

Marinsky, J.A.; Mathuthu, A.; Ephraim, J.H.; Reddy, M.M.

1999-01-01

Calcium ion binding to a soil fulvic acid (Armadale Bh Horizon) was evaluated over a range of calcium ion concentrations, from pH 3.8 to 7.3, using potentiometric titrations and calcium ion electrode measurements. Fulvic acid concentration was constant (100 milligrams per liter) and calcium ion concentration varied up to 8 X 10-4 moles per liter. Experiments discussed here included: (1) titrations of fulvic acid-calcium ion containing solutions with sodium hydroxide; and (2) titrations of fully neutralized fulvic acid with calcium chloride solutions. Apparent binding constants (expressed as the logarithm of the value, log ??app) vary with solution pH, calcium ion concentration, degree of acid dissociation, and ionic strength (from log ??app = 2.5 to 3.9) and are similar to those reported by others. Fulvic acid charge, and the associated Donnan Potential, influences calcium ion-fulvic acid ion pair formation. A Donnan Potential corrrection term allowed calculation of intrinsic calcium ion-fulvic acid binding constants. Intrinsic binding constants vary from 1.2 to 2.5 (the average value is about log??= 1.6) and are similar to, but somewhat higher than, stability constants for calcium ion-carboxylic acid monodentate complexes. ?? by Oldenbourg Wissenschaftsverlag, Mu??nchen.

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

PubMed

Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

2016-12-15

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell wall reactivity of acidophilic and alkaliphilic bacteria determined by potentiometric titrations and Cd adsorption experiments.

PubMed

Kenney, Janice P L; Fein, Jeremy B

2011-05-15

In this study, we used potentiometric titrations and Cd adsorption experiments to determine the binding capacities of two acidophilic (A. cryptum and A. acidophilum) and two alkaliphilic (B. pseudofirmus and B. circulans) bacterial species in order to determine if any consistent trends could be observed relating bacterial growth environment to proton and Cd binding properties and to compare those binding behaviors to those of neutrophilic bacteria. All of the bacterial species studied exhibited significant proton buffering over the pH range in this study, with the alkaliphiles exhibiting significantly higher acidity constants than the acidophiles as well as the neutrophilic bacterial consortia. The calculated average site concentrations for each of the bacteria in this study are within 2σ experimental error of each other, with the exception of A. cryptum, which has a significantly higher Site 2 concentration than the other species. Despite differing acidity constants between the acidophiles and alkaliphiles, all bacteria except A. cryptum exhibited remarkably similar Cd adsorption behavior to each other, and the observed extent of adsorption was also similar to that predicted from a generalized model derived using neutrophilic bacterial consortia. This study demonstrates that bacteria that grow under extreme conditions exhibit similar proton and metal adsorption behavior to that of previously studied neutrophilic species and that a single set of proton and metal binding constants can be used to model the behavior of bacterial adsorption under a wide range of environmental conditions.

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

Anion binding by bambus[6]uril probed in the gas phase and in solution.

PubMed

Révész, Agnes; Schröder, Detlef; Svec, Jan; Wimmerová, Michaela; Sindelar, Vladimir

2011-10-20

Electrospray ionization mass spectrometry (ESI-MS) is used to probe the binding of small anions to the macrocycle of bambus[6]uril. For the halide ions, the experimental patterns suggest F(-) < Cl(-) < Br(-) < I(-), which is consistent with the order of anion binding found in the condensed phase. Parallel equilibrium studies in the condensed phase establish the association constants of halide anions and bambus[6]uril in mixed solvents. A detailed analysis of the mass spectrometric data is used to shed light on the correlations between the binding constants in the condensed phase and the ion abundances observed using ESI-MS. From the analysis it becomes apparent that ESI-MS can indeed represent the situation in solution to some extent, but the sampling in the gas-phase experiment is not 1:1 compared to that in solution.

Interaction of milk whey protein with common phenolic acids

NASA Astrophysics Data System (ADS)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Complexes of polyadenylic acid and the methyl esters of amino acids

NASA Technical Reports Server (NTRS)

Khaled, M. A.; Mulins, D. W., Jr.; Swindle, M.; Lacey, J. C., Jr.

1983-01-01

A study of amino acid methyl esters binding to polyadenylic acid supports the theory that the genetic code originated through weak but selective affinities between amino acids and nucleotides. NMR, insoluble complex analysis, and ultraviolet spectroscopy are used to illustrate a correlation between the hydrophybicities of A amino acids and their binding constants, which, beginning with the largest, are in the order of Phe (having nominally a hydrophobic AAA anticodon), Ile, Leu, Val and Gly (having a hydrophilic anticodon with no A). In general, the binding constants are twice the values by Reuben and Polk (1980) for monomeric AMP, which suggests that polymer amino acids are interacting with only one base. No real differences are found betwen poly A binding for free Phe, Phe methyl ester or Phe amide, except that the amide value is slightly lower.

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

PubMed Central

Yuan, Ji; Chow, Dar-Chone; Huang, Wanzhi; Palzkill, Timothy

2011-01-01

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 residue positions in BLIP that contact β-lactamase were randomized and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected and it was found to inhibit the PC1 β-lactamase with a Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme where it forms a salt bridge with BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants. PMID:21238457

Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

NASA Astrophysics Data System (ADS)

Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

2016-03-01

The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

Interaction of the dietary pigment curcumin with hemoglobin: energetics of the complexation.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2014-08-01

Thermodynamics of the interaction of the chemotherapeutic and chemopreventive dietary pigment, curcumin, with hemoglobin was studied by isothermal titration calorimetry. The binding was characterized to be exothermic. At 293.15 K, the equilibrium constant for curcumin-Hb complexation was found to be (4.88 ± 0.06) × 10(5) M(-1). The binding stoichiometry was calculated to be 1.08 ± 0.05, confirming a 1:1 complexation. The binding was driven by a large negative standard molar enthalpy change (ΔH(0) = -118.45 ± 0.05 kJ mol(-1)) and an unfavorable standard molar entropy change (TΔS(0) = -86.53 ± 0.01 kJ mol(-1)) at 293.15 K. Increasing the temperature favoured the binding, and the magnitude of the negative standard molar heat capacity change suggested the involvement of significant hydrophobic forces in the binding process. With increasing salt concentration, the magnitude of the equilibrium constant decreased slightly; and the complexation mostly involved non-polyelectrolytic forces contributing about 92-94% of the standard molar Gibbs energy change. DSC studies revealed that curcumin binding caused a partial unfolding of the protein.

Studies on binding mechanism between carotenoids from sea buckthorn and thermally treated α-lactalbumin

NASA Astrophysics Data System (ADS)

Dumitraşcu, Loredana; Ursache, Florentina Mihaela; Stănciuc, Nicoleta; Aprodu, Iuliana

2016-12-01

Sea buckthorn is a natural food ingredient rich in bioactive compounds such as carotenoids, tocopherols, sterols, flavonoids, lipids, vitamins, tannins and minerals. Herein, fluorescence and UV-vis techniques were used to study the interaction of heat treated α-lactalbumin (α-LA) with carotenoids from sea buckthorn berries extract (CSB) and β-carotene. Further atomic level details on the interaction between α-LA and β-carotene were obtained by means of molecular modelling techniques. The quenching rate constants, binding constants, and number of binding sites were calculated in the presence of CSB. The emission spectral studies revealed that, CSB have the ability to bind α-LA and form a ground state complex via static quenching process. Maximum degree of quenching was reached at 100 °C, where β-carotene and CSB quenched the Trp fluorescence of α-LA by 56% and 47%, respectively. In order to reveal the interaction between CSB and α-LA, the thermodynamic parameters were determined from the van't Hoff plot based on the temperature dependence of the binding constant. In agreement with the in silico observations, the thermodynamic parameters enabled us to consider that the association between α-LA and β-carotene is a spontaneous process driven by enthalpy, dominated mainly by the van der Waals interaction, but hydrophobic interactions might also be considered. The interaction between CSB and α-LA was further confirmed by UV-vis absorption spectra, where a blue shift of position was noticed at higher temperature suggesting the complex formation. The results provided here supply a better understanding of the binding of CSB to α-LA, which can be further exploited in designing new healthy food applications.

Interaction between a cationic porphyrin and ctDNA investigated by SPR, CV and UV-vis spectroscopy.

PubMed

Xu, Zi-Qiang; Zhou, Bo; Jiang, Feng-Lei; Dai, Jie; Liu, Yi

2013-10-01

The interaction between ctDNA and a cationic porphyrin was studied in this work. The binding process was monitored by surface plasmon resonance (SPR) spectroscopy in detail. The association, dissociation rate constants and the binding constants calculated by global analysis were 2.4×10(2)±26.4M(-1)s(-1), 0.011±0.0000056s(-1) and 2.18×10(4)M(-1), respectively. And the results were confirmed by cyclic voltammetry and UV-vis absorption spectroscopy. The binding constants obtained from cyclic voltammetry and UV-vis absorption spectroscopy were 8.28×10(4)M(-1) and 6.73×10(4)M(-1) at 298K, respectively. The covalent immobilization methodology of ctDNA onto gold surface modified with three different compounds was also investigated by SPR. These compounds all contain sulfydryl but with different terminated functional groups. The results indicated that the 11-MUA (HS(CH2)10COOH)-modified gold film is more suitable for studying the DNA-drug interaction. Copyright © 2013 Elsevier B.V. All rights reserved.

Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

NASA Astrophysics Data System (ADS)

Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

2018-01-01

Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein. Characterization and regulation by uridine and guanosine nucleotides

PubMed Central

Jørgensen, Casper Møller; Fields, Christopher J.; Chander, Preethi; Watt, Desmond; Burgner, John W.; Smith, Janet L.; Switzer, Robert L.

2011-01-01

Summary The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5’ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. Apparent dissociation constants at 0° C ranged from 0.13 to 0.87 nM in the absence of effectors; dissociation constants were decreased by 3 to 12 fold by uridine nucleotides and increased by 40 to 200 fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. PMID:18190533

Synthesis and characterization of a new zinc(II) complex with tetradentate azo-thioether ligand: X-ray structure, DNA binding study and DFT calculation

NASA Astrophysics Data System (ADS)

Mondal, Apurba Sau; Pramanik, Ajoy Kumar; Patra, Lakshman; Manna, Chandan Kumar; Mondal, Tapan Kumar

2017-10-01

A new zinc(II) complex, [Zn(L)(H2O)](ClO4) (1) with azo-thioether containing NSNO donor ligand, 3-(2-(2-((pyridin-2-ylmethyl)thio)phenyl)hydrazono)pentane-2,4-dione (HL) is synthesized and characterized by several spectroscopic techniques. The distorted square based pyramidal (DSBP) geometry is confirmed by single crystal X-ray structure. The ability of the complex to bind with CT DNA is investigated by UV-vis method and the binding constant is found to be 4.16 × 104 M-1. Competitive binding study with ethidium bromide (EB) by fluorescence method suggests that the zinc(II) complex efficiently displaces EB from EB-DNA. The Stern-Volmer dynamic quenching constant, Ksv is found to be 1.2 × 104 M-1. Theoretical calculations by DFT and TDDFT/CPCM methods are used to interpret the electronic structure and UV-vis spectrum of the complex.

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of complexes between phenethylamine enantiomers and β-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

PubMed

Wahl, Joachim; Furuishi, Takayuki; Yonemochi, Etsuo; Meinel, Lorenz; Holzgrabe, Ulrike

2017-04-01

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, methyl-β-cyclodextrin and 6-O-α-maltosyl-β-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Study on the interactions between toxic nitroanilines and lysozyme by spectroscopic approaches and molecular modeling.

PubMed

Gu, Yunlan; Wang, Yanqing; Zhang, Hongmei

2018-05-05

Being exogenous environmental pollutants, nitroanilines (NAs) are highly toxic and have mutagenic and carcinogenic activity. Being lack of studies on interactions between NAs and lysozyme at molecular level, the binding interactions of lysozyme with o-nitroaniline (oNA), m-nitroaniline (mNA) and p-nitroaniline (pNA) were investigated by means of steady-state fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy, as well as molecular modeling. The experimental results revealed that the fluorescence of lysozyme is quenched by oNA and mNA through a static quenching, while the fluorescence quenching triggered by pNA is a combined dynamic and static quenching. The number of binding sites (n) and the binding constant (K b ) corresponding thermodynamic parameters ΔH ⊖ , ΔS ⊖ , ΔG ⊖ at different temperatures were calculated. The reactions between NAs and lysozyme were spontaneous and entropy driven and the binding of NAs to lysozyme induced conformation changes of lysozyme. The difference of the position of -NO 2 group affected the binding and the binding constants K b decreased in the following pattern: K b (pNA) >K b (mNA) >K b (oNA). Molecular docking studies were performed to reveal the most favorable binding sites of NAs on lysozyme. Our recently results could offer mechanistic insights into the nature of the binding interactions between NAs and lysozyme and provide information about the toxicity risk of NAs to human health. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

PubMed

Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

2015-03-01

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

Proton and metal ion binding to natural organic polyelectrolytes-I. Studies with synthetic model compounds

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

A unified physico-chemical model, based on a modified Henderson-Hasselbalch equation, for the analysis of ion complexation reactions involving charged polymeric systems is presented and verified. In this model pH = pKa+p(??Ka) + log(??/1 - ??) where Ka is the intrinsic acid dissociation constant of the ionizable functional groups on the polymer, ??Ka is the deviation of the intrinsic constant due to electrostatic interaction between the hydrogen ion and the polyanion, and alpha (??) is the polyacid degree of ionization. Using this approach pKa values for repeating acidic units of polyacrylic (PAA) and polymethacrylic (PMA) acids were found to be 4.25 ?? 0.03 and 4.8 ?? 0.1, respectively. The polyion electrostatic deviation term derived from the potentiometric titration data (i.e. p(??Ka)) is used to calculate metal ion concentration at the complexation site on the surface of the polyanion. Intrinsic cobalt-polycarboxylate binding constants (7.5 for PAA and 5.6 for PMA), obtained using this procedure, are consistent with the range of published binding constants for cobalt-monomer carboxylate complexes. In two phase systems incorporation of a Donnan membrane potential term allows determination of the intrinsic pKa of a cross-linked PMA gel, pKa = 4.83, in excellent agreement with the value obtained for the linear polyelectrolyte and the monomer. Similarly, the intrinsic stability constant for cobalt ion binding to a PMA-gel (??CoPMA+ = 11) was found to be in agreement with the linear polyelectrolyte analogue and the published data for cobalt-carboxylate monodentate complexes. ?? 1984.

Using resonance light scattering and UV/vis absorption spectroscopy to study the interaction between gliclazide and bovine serum albumin.

PubMed

Zhang, Qiu-Ju; Liu, Bao-Sheng; Li, Gai-Xia; Han, Rong

2016-08-01

At different temperatures (298, 310 and 318 K), the interaction between gliclazide and bovine serum albumin (BSA) was investigated using fluorescence quenching spectroscopy, resonance light scattering spectroscopy and UV/vis absorption spectroscopy. The first method studied changes in the fluorescence of BSA on addition of gliclazide, and the latter two methods studied the spectral change in gliclazide while BSA was being added. The results indicated that the quenching mechanism between BSA and gliclazide was static. The binding constant (Ka ), number of binding sites (n), thermodynamic parameters, binding forces and Hill's coefficient were calculated at three temperatures. Values for the binding constant obtained using resonance light scattering and UV/vis absorption spectroscopy were much greater than those obtained from fluorescence quenching spectroscopy, indicating that methods monitoring gliclazide were more accurate and reasonable. In addition, the results suggest that other residues are involved in the reaction and the mode 'point to surface' existed in the interaction between BSA and gliclazide. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Assessment of amsacrine binding with DNA using UV-visible, circular dichroism and Raman spectroscopic techniques.

PubMed

Jangir, Deepak Kumar; Dey, Sanjay Kumar; Kundu, Suman; Mehrotra, Ranjana

2012-09-03

Proper understanding of the mechanism of binding of drugs to their targets in cell is a fundamental requirement to develop new drug therapy regimen. Amsacrine is a rationally designed anticancer drug, used to treat leukemia and lymphoma. Binding with cellular DNA is a crucial step in its mechanism of cytotoxicity. Despite numerous studies, DNA binding properties of amsacrine are poorly understood. Its reversible binding with DNA does not permit X-ray crystallography or NMR spectroscopic evaluation of amsacrine-DNA complexes. In the present work, interaction of amsacrine with calf thymus DNA is investigated at physiological conditions. UV-visible, FT-Raman and circular dichroism spectroscopic techniques were employed to determine the binding mode, binding constant, sequence specificity and conformational effects of amsacrine binding to native calf thymus DNA. Our results illustrate that amsacrine interacts with DNA by and large through intercalation between base pairs. Binding constant of the amsacrine-DNA complex was found to be K=1.2±0.1×10(4) M(-1) which is indicative of moderate type of binding of amsacrine to DNA. Raman spectroscopic results suggest that amsacrine has a binding preference of intercalation between AT base pairs of DNA. Minor groove binding is also observed in amsacrine-DNA complexes. These results are in good agreement with in silico investigation of amsacrine binding to DNA and thus provide detailed insight into DNA binding properties of amsacrine, which could ultimately, renders its cytotoxic efficacy. Copyright © 2012 Elsevier B.V. All rights reserved.

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

PubMed

Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per

2015-08-04

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching, UV/vis and FTIR spectroscopic techniques.

PubMed

Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

2016-03-01

The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non-fluorescent CF-CFA and CF-CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (KSV), quenching rate constant (kq), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (KSV) between CF and CGA, CFA are 1.84 × 10(4) and 1.04 × 10(4) L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA. Copyright © 2015 John Wiley & Sons, Ltd.

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

PubMed

Salaniwal, Sumeet; Manas, Eric S; Alvarez, Juan C; Unwalla, Rayomand J

2007-02-01

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method. Copyright 2006 Wiley-Liss, Inc.

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Metal binding characterization and conformational studies using Raman microscopy of resin-bound poly(aspartic acid).

PubMed

Stair, Jacqueline L; Holcombe, James A

2007-03-01

The metal binding capacities, conditional stability constants, and secondary structure of immobilized polyaspartic acid (PLAsp) (n = 6, 20, and 30) on TentaGel resin were determined when binding Mg2+, Co2+, Cd2+, and Ni2+. Metal binding to the synthesized peptides was evaluated using breakthrough curves from a packed microcolumn and flame atomic absorption spectrophotometry (FAAS) detection. The metal capacities reached values of 590, 2160, and 3710 mumol of metal/g of resin for the 6-mer, 20-mer, and 30-mer, respectively, and this resulted in 2-3 residues per metal for all peptides and metals tested. Surprisingly, the concentrated environment of the resin along with the spatial distribution of attachment groups allowed for most residues to participate in metal binding regardless of the peptide length. Conditional stability constants calculated using single metal binding isotherms indicated that binding strength decreased as the chain length increased on the resin. Raman microscopy on single beads was used to determine PLAsp secondary structure, and all peptides were of a mixed conformation (i.e., beta-sheets, alpha-helices, random chain, etc.) during neutral conditioning and metal binding. Uniquely, the longer 20-mer and 30-mer peptides showed a distinct change from a mixed conformation to beta-sheets and alpha-helices during metal release with acid. This study confirms that metal release by longer immobilized peptides is often assisted by a conformational change, which easily spoils the binding cavity, while shorter peptides may release metal primarily by H+ displacement.

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods.

PubMed

Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

2017-08-01

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10 10  L mol -1  s -1 , indicating forming QNPL-BSA complex through the intermolecular binding interaction. The binding constant for the QNPL-BSA complex is in the order of 10 5  M -1 , indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal's forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

Rationalizing 5000-Fold Differences in Receptor-Binding Rate Constants of Four Cytokines

PubMed Central

Pang, Xiaodong; Qin, Sanbo; Zhou, Huan-Xiang

2011-01-01

The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as ka=ka0exp(−ΔGel∗/kBT) where ka0 is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (ka0) of EPO, IL4, hGH, and PRL were similar (5.2 × 105 M−1s−1, 2.4 × 105 M−1s−1, 1.7 × 105 M−1s−1, and 1.7 × 105 M−1s−1, respectively). However, the average electrostatic free energies (ΔGe1∗) were very different (−4.2 kcal/mol, −2.4 kcal/mol, −0.1 kcal/mol, and −0.5 kcal/mol, respectively, at ionic strength = 160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 108 M−1s−1, 1.3 × 107 M−1s−1, 2.0 × 105 M−1s−1, and 7.6 × 104 M−1s−1, respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps evolutionarily tuned. Our calculations also reproduced well-observed effects of mutations and ionic strength on the rate constants and produced a set of mutations on the complex of hGH with its receptor that putatively enhances the rate constant by nearly 100-fold through increasing charge complementarity. To quantify charge complementarity, we propose a simple index based on the charge distribution within the binding interface, which shows good correlation with ΔGe1∗. Together these results suggest that protein charges can be manipulated to tune ka and control biological function. PMID:21889455

Study of DNA-emodin interaction by FTIR and UV-vis spectroscopy.

PubMed

Saito, Samuel T; Silva, Givaldo; Pungartnik, Cristina; Brendel, Martin

2012-06-04

Emodin, a plant- and fungus-derived anthraquinone, exerts genotoxic and antioxidative effects and shows promise in antitumor and antibacterial therapies. The aim of this study was to examine the molecular interactions of emodin with DNA in aqueous solution at physiological pH using spectroscopic methods. Fourier Transform Infrared (FTIR) Spectroscopy and UV absorption spectra were used to determine the structural features, the binding mode and the association constants. Our UV-spectroscopic results indicate that emodin interacts with DNA by intercalation and by external binding. FTIR results suggest that emodin interaction occurs preferably via adenine and thymine base pairs and also weakly with the phosphate backbone of the DNA double helix. The binding constant for emodin-DNA complex formation is estimated to be K=5.59×10(3)M(-1). No significant changes of DNA conformation were observed upon emodin-DNA complexation. Copyright © 2012 Elsevier B.V. All rights reserved.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors.

PubMed

Jadey, Snehal; Auerbach, Anthony

2012-07-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes ("catch" and "hold") that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement ("capping"). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

PubMed Central

Jadey, Snehal

2012-01-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation. PMID:22732309

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise

PubMed Central

1995-01-01

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed. PMID:7539481

Metal adsorption onto bacterial surfaces: development of a predictive approach

NASA Astrophysics Data System (ADS)

Fein, Jeremy B.; Martin, Aaron M.; Wightman, Peter G.

2001-12-01

Aqueous metal cation adsorption onto bacterial surfaces can be successfully modeled by means of a surface complexation approach. However, relatively few stability constants for metal-bacterial surface complexes have been measured. In order to determine the bacterial adsorption behavior of cations that have not been studied in the laboratory, predictive techniques are required that enable estimation of the stability constants of bacterial surface complexes. In this study, we use a linear free-energy approach to compare previously measured stability constants for Bacillus subtilis metal-carboxyl surface complexes with aqueous metal-organic acid anion stability constants. The organic acids that we consider are acetic, oxalic, citric, and tiron. We add to this limited data set by conducting metal adsorption experiments onto Bacillus subtilis, determining bacterial surface stability constants for Co, Nd, Ni, Sr, and Zn. The adsorption behavior of each of the metals studied here was described well by considering metal-carboxyl bacterial surface complexation only, except for the Zn adsorption behavior, which required carboxyl and phosphoryl complexation to obtain a suitable fit to the data. The best correlation between bacterial carboxyl surface complexes and aqueous organic acid anion stability constants was obtained by means of metal-acetate aqueous complexes, with a linear correlation coefficient of 0.97. This correlation applies only to unhydrolyzed aqueous cations and only to carboxyl binding of those cations, and it does not predict the binding behavior under conditions where metal binding to other bacterial surface site types occurs. However, the relationship derived in this study permits estimation of the carboxyl site adsorption behavior of a wide range of aqueous metal cations for which there is an absence of experimental data. This technique, coupled with the observation of similar adsorption behaviors across bacterial species (Yee and Fein, 2001), enables estimation of the effects of bacterial adsorption on metal mobilities for a large number of environmental and geologic applications.

Spectral Study of the Interaction of Myoglobin with Tannin

NASA Astrophysics Data System (ADS)

Grigoryan, K. R.; Sargsyan, L. S.

2016-07-01

The interaction of myoglobin with tannin (tannic acid) at 298.15 and 303.15 K was studied by fluorescence and absorption spectroscopy in the UV region. The physicochemical and thermodynamic binding parameters (the fluorescence quenching mechanism, the bonding constant, the number of binding sites, the type of interaction) and parameters of the formed complex were determined. It was found that binding of myoglobin with tannic acid does not lead to significant changes in the electronic state of the heme ring of myoglobin.

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.

The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry

PubMed Central

Sun, Lijun; Gidley, Michael J.

2017-01-01

Scope This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA). Methods and results The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K ic), quenching constant (K FQ) and binding constant (K itc). Conclusion The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. PMID:28618113

Interaction of an antiepileptic drug, lamotrigine with human serum albumin (HSA): Application of spectroscopic techniques and molecular modeling methods.

PubMed

Poureshghi, Fatemeh; Ghandforoushan, Parisa; Safarnejad, Azam; Soltani, Somaieh

2017-01-01

Lamotrigine (an epileptic drug) interaction with human serum albumin (HSA) was investigated by fluorescence, UV-Vis, FTIR, CD spectroscopic techniques, and molecular modeling methods. Binding constant (K b ) of 5.74×10 3 and number of binding site of 0.97 showed that there is a slight interaction between lamotrigine and HSA. Thermodynamic studies was constructed using the flourimetric titrations in three different temperatures and the resulted data used to calculate the parameters using Vant Hoff equation. Decreased Stern Volmer quenching constant by enhanced temperature revealed the static quenching mechanism. Negative standard enthalpy (ΔH) and standard entropy (ΔS) changes indicated that van der Waals interactions and hydrogen bonds were dominant forces which facilitate the binding of Lamotrigine to HSA, the results were confirmed by molecular docking studies which showed no hydrogen binding. The FRET studies showed that there is a possibility of energy transfer between Trp214 and lamotrigine. Also the binding of lamotrigine to HSA in the studied concentrations was not as much as many other drugs, but the secondary structure of the HSA was significantly changed following the interaction in a way that α-helix percentage was reduced from 67% to 57% after the addition of lamotrigine in the molar ratio of 4:1 to HSA. According to the docking studies, lamotrigine binds to IB site preferably. Copyright © 2016. Published by Elsevier B.V.

In-vitro Equilibrium Phosphate Binding Study of Sevelamer Carbonate by UV-Vis Spectrophotometry.

PubMed

Prasaja, Budi; Syabani, M Maulana; Sari, Endah; Chilmi, Uci; Cahyaningsih, Prawitasari; Kosasih, Theresia Weliana

2018-06-12

Sevelamer carbonate is a cross-linked polymeric amine; it is the active ingredient in Renvela ® tablets. US FDA provides recommendation for demonstrating bioequivalence for the development of a generic product of sevelamer carbonte using in-vitro equilibrium binding study. A simple UV-vis spectrophotometry method was developed and validated for quantification of free phosphate to determine the binding parameter constant of sevelamer. The method validation demonstrated the specificity, limit of quantification, accuracy and precision of measurements. The validated method has been successfully used to analyze samples in in-vitro equilibrium binding study for demonstrating bioequivalence. © Georg Thieme Verlag KG Stuttgart · New York.

New Fluorescent Macrolide Derivatives for Studying Interactions of Antibiotics and Their Analogs with the Ribosomal Exit Tunnel.

PubMed

Tereshchenkov, A G; Shishkina, A V; Karpenko, V V; Chertkov, V A; Konevega, A L; Kasatsky, P S; Bogdanov, A A; Sumbatyan, N V

2016-10-01

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

Determination of the molecular complexation constant between alprostadil and alpha-cyclodextrin by conductometry: implications for a freeze-dried formulation.

PubMed

Sheehy, Philip M; Ramstad, Tore

2005-10-04

The binding constant between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD) was determined at four temperatures using conductance measurements. Alpha-cyclodextrin is an excipient material in Caverject dual chamber syringe (DCS) that was added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, since only the free drug is clinically active. The conductivity measurement is based on a decrease in specific conductance as alprostadil is titrated with alpha-CD. The change in conductivity was plotted versus free ligand concentration (alpha-CD) to generate a binding curve. As the value of the binding constant proved to be dependent on substrate concentration, it is really a pseudo binding constant. A value of 742+/-60 M(-1) was obtained for a 0.5 mM solution of alprostadil at 27 degrees C and a value of 550+/-52 M(-1) at 37 degrees C. These results compare favorably to values previously obtained by NMR and capillary electrophoresis. Calculation of the fraction PGE1 free upon reconstitution and injection show it to approach the desired outcome of one. Hence, the amount of drug delivered by Caverject DCS is nominally equivalent to that delivered by Caverject S. Po., a predecessor product that contains no alpha-cyclodextrin.

Synthesis, characterization and serum albumin binding studies of vitamin K3 derivatives.

PubMed

Suganthi, Murugesan; Elango, Kuppanagounder P

2017-01-01

Synthesis, characterization and bovine serum albumin (BSA) binding properties of three derivatives of vitamin K3 have been described. Results of UV-Vis and fluorescence spectra indicate complexation between BSA and the ligands with conformational changes in protein, which is strongly supported by synchronous and three dimensional fluorescence studies. Addition of the ligands quenches the fluorescence of BSA which is accompanied by reduction in quantum yield (Ф) from 0.1010 to 0.0775-0.0986 range. Thermodynamic investigations reveal that hydrophobic interaction is the major binding force in the spontaneous binding of these ligands with BSA. The binding constants obtained depend on the substituent present in the quinone ring, which correlates linearly with the Taft's field substituent constant (σ F ). The results show that compound with strong electron withdrawing nitro-group forms relatively stronger complex with BSA than amino and thioglycolate substituted ones. Circular dichroism studies show that the α-helical content of the protein, upon complexation with the ligands, decreases in the case of amino and nitro substituted vitamin K3 while increases in thioglycolate substituted compound. Molecular docking studies indicated that the vitamin K3 derivatives are surrounded by hydrophobic residues of the BSA molecule, which is in good agreement with the results of fluorescence spectral and thermodynamic studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Experimental and computational studies on the effects of valganciclovir as an antiviral drug on calf thymus DNA.

PubMed

Shahabadi, Nahid; Pourfoulad, Mehdi; Moghadam, Neda Hosseinpour

2017-01-02

DNA-binding properties of an antiviral drug, valganciclovir (valcyte) was studied by using emission, absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, and computational studies. The drug bound to calf thymus DNA (ct-DNA) in a groove-binding mode. The calculated binding constant of UV-vis, K a , is comparable to groove-binding drugs. Competitive fluorimetric studies with Hoechst 33258 showed that valcyte could displace the DNA-bound Hoechst 33258. The drug could not displace intercalated methylene blue from DNA double helix. Furthermore, the induced detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity confirm the groove-binding mode. In addition, an integrated molecular docking was employed to further investigate the binding interactions between valcyte and calf thymus DNA.

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NASA Astrophysics Data System (ADS)

Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

2012-10-01

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

New cobalt(II) and nickel(II) complexes of benzyl carbazate Schiff bases: Syntheses, crystal structures, in vitro DNA and HSA binding studies.

PubMed

Nithya, Palanivelu; Helena, Sannasi; Simpson, Jim; Ilanchelian, Malaichamy; Muthusankar, Aathi; Govindarajan, Subbiah

2016-12-01

In the present study, new Schiff base complexes with the composition [M(NCS) 2 (L1) 2 ]·nH 2 O, where M=Co (n=0) (1) and Ni (n=2) (2); [M(NCS) 2 (L2) 2 ], M=Co (3) and Ni (4) as well as [M(NCS) 2 (L3) 2 ], M=Co (5) and Ni (6); (L1=benzyl 2-(propan-2-ylidene)hydrazinecarboxylate, L2=benzyl 2-(butan-2-ylidene)hydrazinecarboxylate and L3=benzyl 2-(pentan-3-ylidene)hydrazinecarboxylate) have been synthesized by a template method. The complexes were characterised by analytical methods, spectroscopic studies, thermal and X-ray diffraction techniques. The structures of all the complexes explore that the metal(II) cation has a trans-planar coordination environment, the monomeric units containing a six-coordinated metal center in octahedral geometry with N-bound isothiocyanate anions coordinated as terminal ligands. Furthermore, the binding of the two Schiff base ligands to the metal centers involves the azomethine nitrogen and the carbonyl oxygen in mutually trans configuration. The binding interactions of all the complexes with Calf thymus-deoxyribonucleic acid (CT-DNA) and human serum albumin (HSA) have been investigated using absorption and emission spectral techniques. The CT-DNA binding properties of these complexes reveal that they bind to CT-DNA through a partial intercalation mode and the binding constant values were calculated using the absorption and emission spectral data. The binding constant values (~10×10 6 moldm -3 ) indicate strong binding of metal complexes with CT-DNA. HSA binding interaction studies showed that the cobalt and nickel complexes can quench the intrinsic fluorescence of HSA through static quenching process. Also, molecular docking studies were supported out to apprehend the binding interactions of these complexes with DNA and HSA which offer new understandings into the experimental model observations. Copyright © 2016 Elsevier B.V. All rights reserved.

[Binding studies with Ulex europaeus agglutinin I (UEA-I) of the vascular endothelium of the synovial membrane].

PubMed

Zschäbitz, A; Stofft, E

1988-01-01

The lectin binding sites of the synovium of patients with rheumatoid arthritis and osteoarthritis were investigated. It was shown that Ulex europaeus agglutinin is a constant marker of the vascular endothelium and is not induced during the course of inflammatory process in rheumatoid arthritis.

Studies on the interactions of 3,6-diaminoacridine derivatives with human serum albumin by fluorescence spectroscopy.

PubMed

Gökoğlu, Elmas; Kıpçak, Fulya; Seferoğlu, Zeynel

2014-11-01

This study reports the preparation and investigation of the modes of binding of the two symmetric 3,6-diaminoacridine derivatives obtained from proflavine, which are 3,6-diphenoxycarbonyl aminoacridine and 3,6-diethoxycarbonyl aminoacridine to human serum albumin (HSA). The interaction of HSA with the derivatives was investigated using fluorescence quenching and ultraviolet-visible absorption spectra at pH 7.2 and different temperatures. The results suggest that the derivatives used can interact strongly with HSA and are the formation of HSA-derivative complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern-Volmer quenching constants, binding constants, binding sites and corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The binding distance (r) ~ 3 nm between the donor (HSA) and acceptors (3,6-diethoxycarbonyl aminoacridine, 3,6-diphenoxycarbonyl aminoacridine and proflavine) was obtained according to Förster's non-radiative energy transfer theory. Moreover, the limit of detection and limit of quantification of derivatives were calculated in the presence of albumin. Copyright © 2014 John Wiley & Sons, Ltd.

CONSTANTS FOR MERCURY BINDING BY DISSOLVED ORGANIC MATTER ISOLATES FROM THE FLORIDA EVERGLADES. (R827653)

EPA Science Inventory

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand appro...

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis

NASA Astrophysics Data System (ADS)

Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

2016-09-01

The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb = (7.6 ± 0.21) × 105) between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2 ± 0.11) × 106 M- 1. Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

Misuse of thermodynamics in the interpretation of isothermal titration calorimetry data for ligand binding to proteins.

PubMed

Pethica, Brian A

2015-03-01

Isothermal titration calorimetry (ITC) has given a mass of data on the binding of small molecules to proteins and other biopolymers, with particular interest in drug binding to proteins chosen as therapeutic indicators. Interpretation of the enthalpy data usually follows an unsound protocol that uses thermodynamic relations in circumstances where they do not apply. Errors of interpretation include incomplete definitions of ligand binding and equilibrium constants and neglect of the non-ideality of the solutions under study, leading to unreliable estimates of standard free energies and entropies of binding. The mass of reported thermodynamic functions for ligand binding to proteins estimated from ITC enthalpies alone is consequently of uncertain thermodynamic significance and utility. ITC and related experiments to test the protocol assumptions are indicated. A thermodynamic procedure avoiding equilibrium constants or other reaction models and not requiring protein activities is given. The discussion draws attention to the fundamental but neglected relation between the thermodynamic activity and bioactivity of drugs and to the generally unknown thermodynamic status of ligand solutions, which for drugs relates directly to effective therapeutic dosimetry. Copyright © 2014 Elsevier Inc. All rights reserved.

Preliminary selection and evaluation of the binding of aptamers against a Hantavirus antigen using fluorescence spectroscopy and modeling

NASA Astrophysics Data System (ADS)

Missailidis, Sotiris; de Oliveira, Renata Carvalho; Silva, Dilson; Cortez, Célia Martins; Guterres, Alexandro; Vicente, Luciana Helena Bassan; de Godoy, Daniela Tupy; Lemos, Elba

2015-12-01

In this study we have aimed to develop novel aptamers against the Hantavirus nucleoprotein N, a valid antigen already used in the Hantavirus reference laboratory of the Institute Oswaldo Cruz in Rio de Janeiro, Brazil. Such aptamers, if they are found to bind with high affinity and specificity for the selected hantavirus antigen, they could be translated into novel diagnostic assays with the ability to provide early detection for hantaviroses and their related disease syndromes. In a preliminary screening, we have managed to identify three aptamer species. We have analyzed a short and a long version of these aptamer using fluorescence spectroscopy and modelled their binding. We have identified Stern-Volmer constants for the selected aptamers, which have shown affinity for their target, with a different binding between the short and the long versions of them. Short aptamers have shown to have a higher Stern-Volmer constant and the ability to potentially bind to more than one binding site on the antigen. The information provided by the spectroscopic screening has been invaluable in allowing us to define candidates for further development into diagnostic assays.

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

PubMed

Weikl, Thomas R; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-09-02

The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

PubMed Central

Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-01-01

ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

PubMed

Dobreva, Marina A; Green, Rebecca J; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Howlin, Brendan J; Frazier, Richard A

2014-09-17

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV).

DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin.

PubMed

Kanakis, C D; Tarantilis, P A; Polissiou, M G; Diamantoglou, S; Tajmir-Riahi, H A

2005-06-01

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants K(que) = 7.25 x 10(4)M(-1), K(kae) = 3.60 x 10(4)M(-1), and K(del) = 1.66 x 10(4)M(-1). The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.

Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

2007-03-01

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

PubMed

Le Saux, Thomas; Hisamoto, Hideaki; Terabe, Shigeru

2006-02-03

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Kinetic modeling of benzodiazepine receptor binding with PET and high specific activity [(11)C]Iomazenil in healthy human subjects.

PubMed

Bremner, J D; Horti, A; Staib, L H; Zea-Ponce, Y; Soufer, R; Charney, D S; Baldwin, R

2000-01-01

Quantitation of the PET benzodiazepine receptor antagonist, [(11)C]Iomazenil, using low specific activity radioligand was recently described. The purpose of this study was to quantitate benzodiazepine receptor binding in human subjects using PET and high specific activity [(11)C]Iomazenil. Six healthy human subjects underwent PET imaging following a bolus injection of high specific activity (>100 Ci/mmol) [(11)C]iomazenil. Arterial samples were collected at multiple time points after injection for measurement of unmetabolized total and nonprotein-bound parent compound in plasma. Time activity curves of radioligand concentration in brain and plasma were analyzed using two and three compartment model. Kinetic rate constants of transfer of radioligand between plasma, nonspecifically bound brain tissue, and specifically bound brain tissue compartments were fitted to the model. Values for fitted kinetic rate constants were used in the calculation of measures of benzodiazepine receptor binding, including binding potential (the ratio of receptor density to affinity), and product of BP and the fraction of free nonprotein-bound parent compound (V(3)'). Use of the three compartment model improved the goodness of fit in comparison to the two compartment model. Values for kinetic rate constants and measures of benzodiazepine receptor binding, including BP and V(3)', were similar to results obtained with the SPECT radioligand [(123)I]iomazenil, and a prior report with low specific activity [(11)C]Iomazenil. Kinetic modeling using the three compartment model with PET and high specific activity [(11)C]Iomazenil provides a reliable measure of benzodiazepine receptor binding. Synapse 35:68-77, 2000. Published 2000 Wiley-Liss, Inc.

Eu(III) complexes as Anion-responsive Luminescent Sensors and PARACEST Agents

PubMed Central

Hammell, Jacob; Buttarazzi, Leandro; Huang, Ching-Hui; Morrow, Janet R.

2011-01-01

The Eu(III) complex of (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (S-THP) is studied as a sensor for biologically relevant anions. Anion interactions produce changes in the luminescence emission spectrum of the Eu(III) complex, in the 1H NMR spectrum, and correspondingly, in the PARACEST spectrum of the complex (PARACEST = paramagnetic chemical exchange saturation transfer). Direct excitation spectroscopy and luminescence lifetime studies of Eu(S-THP) give information about the speciation and nature of anion interactions including carbonate, acetate, lactate, citrate, phosphate and methylphosphate at pH 7.2. Data is consistent with the formation of both innersphere and outersphere complexes of Eu(S-THP) with acetate, lactate and carbonate. These anions have weak dissociation constants that range from 19–38 mM. Citrate binding to Eu(S-THP) is predominantly innersphere with a dissociation constant of 17 μM. Luminescence emission peak changes upon addition of anion to Eu(S-THP) show that there are two distinct binding events for phosphate and methylphosphate with dissociation constants of 0.3 mM and 3.0 mM for phosphate and 0.6 mM and 9.8 mM for methyl phosphate. Eu(THPC) contains an appended carbostyril derivative as an antenna to sensitize Eu(III) luminescence. Eu(THPC) binds phosphate and citrate with dissociation constants that are 10-fold less than that of the Eu(S-THP) parent, suggesting that functionalization through a pendent group disrupts the anion binding site. Eu(S-THP) functions as an anion responsive PARACEST agent through exchange of the alcohol protons with bulk water. The alcohol proton resonances of Eu(S-THP) shift downfield in the presence of acetate, lactate, citrate and methylphosphate, giving rise to distinct PARACEST peaks. In contrast, phosphate binds to Eu(S-THP) to suppress the PARACEST alcohol OH peak and carbonate does not markedly change the alcohol peak at 5 mM Eu(S-THP), 15 mM carbonate at pH 6.5 or 7.2. This work shows that the Eu(S-THP) complex has unique selectivity toward binding of biologically relevant anions and that anion binding results in changes in both the luminescence and PARACEST spectra of the complex. PMID:21548563

Eu(III) complexes as anion-responsive luminescent sensors and paramagnetic chemical exchange saturation transfer agents.

PubMed

Hammell, Jacob; Buttarazzi, Leandro; Huang, Ching-Hui; Morrow, Janet R

2011-06-06

The Eu(III) complex of (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (S-THP) is studied as a sensor for biologically relevant anions. Anion interactions produce changes in the luminescence emission spectrum of the Eu(III) complex, in the (1)H NMR spectrum, and correspondingly, in the PARACEST spectrum of the complex (PARACEST = paramagnetic chemical exchange saturation transfer). Direct excitation spectroscopy and luminescence lifetime studies of Eu(S-THP) give information about the speciation and nature of anion interactions including carbonate, acetate, lactate, citrate, phosphate, and methylphosphate at pH 7.2. Data is consistent with the formation of both innersphere and outersphere complexes of Eu(S-THP) with acetate, lactate, and carbonate. These anions have weak dissociation constants that range from 19 to 38 mM. Citrate binding to Eu(S-THP) is predominantly innersphere with a dissociation constant of 17 μM. Luminescence emission peak changes upon addition of anion to Eu(S-THP) show that there are two distinct binding events for phosphate and methylphosphate with dissociation constants of 0.3 mM and 3.0 mM for phosphate and 0.6 mM and 9.8 mM for methyl phosphate. Eu(THPC) contains an appended carbostyril derivative as an antenna to sensitize Eu(III) luminescence. Eu(THPC) binds phosphate and citrate with dissociation constants that are 10-fold less than that of the Eu(S-THP) parent, suggesting that functionalization through a pendent group disrupts the anion binding site. Eu(S-THP) functions as an anion responsive PARACEST agent through exchange of the alcohol protons with bulk water. The alcohol proton resonances of Eu(S-THP) shift downfield in the presence of acetate, lactate, citrate, and methylphosphate, giving rise to distinct PARACEST peaks. In contrast, phosphate binds to Eu(S-THP) to suppress the PARACEST alcohol OH peak and carbonate does not markedly change the alcohol peak at 5 mM Eu(S-THP), 15 mM carbonate at pH 6.5 or 7.2. This work shows that the Eu(S-THP) complex has unique selectivity toward binding of biologically relevant anions and that anion binding results in changes in both the luminescence and the PARACEST spectra of the complex. © 2011 American Chemical Society

Synthesis of a zinc(II) complex with hexadentate N4S2 donor thioether ligand: X-ray structure, DNA binding study and DFT computation

NASA Astrophysics Data System (ADS)

Mondal, Apurba Sau; Jana, Mahendra Sekhar; Manna, Chandan Kumar; Naskar, Rahul; Mondal, Tapan Kumar

2018-07-01

A new zinc(II) complex, [Zn(L)](ClO4) with hexadentate N4S2 donor azo-thioether ligand (HL) was synthesized and characterized by several spectroscopic techniques. The structure was confirmed by single crystal X-ray analysis. The interaction of the complex with CT DNA was investigated by UV-vis method and binding constant is found to be 6.6 × 104 M-1. Competitive binding titration with ethidium bromide (EB) by fluorescence titration method reveals that the complex efficiently displaces EB from EB-DNA system and the Stern-Volmer dynamic quenching constant, Ksv is found to be 2.6 × 104 M-1. DFT and TDDFT calculations were carried out to interpret the electronic structure and electronic spectra of the complex.

Pressure-induced increase of exciton-LO-phonon coupling in a ZnCdSe/ZnSe quantum well

NASA Astrophysics Data System (ADS)

Guo, Z. Z.; Liang, X. X.; Ban, S. L.

2003-07-01

The possibility of pressure-induced increase of exciton-LO-phonon coupling in ZnCdSe/ZnSe quantum wells is studied. The ground state binding energies of the heavy hole excitons are calculated using a variational method with consideration of the electron-phonon interaction and the pressure dependence of the parameters. The results show that for quantum wells with intermediate well width, the exciton binding energy and the LO-phonon energy may coincide in the course of pressure increasing, resulting in the increase of exciton-LO-phonon coupling. It is also found that among the pressure-dependent parameters, the influence of the lattice constant is the most important one. The changes of both the effective masses and the dielectric constants have obvious effects on the exciton binding energy, but their influences are counterbalanced.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

PubMed

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

2014-01-03

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. Copyright © 2013 Elsevier Inc. All rights reserved.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

PubMed Central

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B.; Erman, James E.

2013-01-01

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M−1s−1 and 0.34 ± 0.15 s−1, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min−1 at pH 6.0. PMID:24291498

NMR diffusion and relaxation studies of 2-nitroimidazole and albumin interactions

NASA Astrophysics Data System (ADS)

Wijesekera, Dj; Willis, Scott A.; Gupta, Abhishek; Torres, Allan M.; Zheng, Gang; Price, William S.

2018-03-01

Nitroimidazole derivatives are of current interest in the development of hypoxia targeting agents and show potential in the establishment of quantitative measures of tumor hypoxia. In this study, the binding of 2-nitroimidazole to albumin was probed using NMR diffusion and relaxation measurements. Binding studies were conducted at three different protein concentrations (0.23, 0.30 and 0.38 mM) with drug concentrations ranging from 0.005-0.16 M at 298 K. Quantitative assessments of the binding model were made by evaluating the number of binding sites, n, and association constant, K. These were determined to be 21 ± 3 and 53 ± 4 M- 1, respectively.

Stable nanoconjugates of transferrin with alloyed quaternary nanocrystals Ag-In-Zn-S as a biological entity for tumor recognition.

PubMed

Matysiak-Brynda, Edyta; Bujak, Piotr; Augustin, Ewa; Kowalczyk, Agata; Mazerska, Zofia; Pron, Adam; Nowicka, Anna M

2018-01-18

One way to limit the negative effects of anti-tumor drugs on healthy cells is targeted therapy employing functionalized drug carriers. Here we present a biocompatible and stable nanoconjugate of transferrin anchored to Ag-In-Zn-S quantum dots modified with 11-mercaptoundecanoic acid (Tf-QD) as a drug carrier versus typical anticancer drug, doxorubicin. Detailed investigations of Tf-QD nanoconjugates without and with doxorubicin by fluorescence studies and cytotoxic measurements showed that the biological activity of both the transferrin and doxorubicin was fully retained in the nanoconjugate. In particular, the intercalation capabilities of free doxorubicin versus ctDNA remained essentially intact upon its binding to the nanoconjugate. In order to evaluate these capabilities, we studied the binding constant of doxorubicin attached to Tf-QDs with ctDNA as well as the binding site size on the ctDNA molecule. The binding constant slightly decreased compared to that of free doxorubicin while the binding site size, describing the number of consecutive DNA lattice residues involved in the binding, increased. It was also demonstrated that the QDs alone and in the form of a nanoconjugate with Tf were not cytotoxic towards human non-small cell lung carcinoma (H460 cell line) and the tumor cell sensitivity of the DOX-Tf-QD nanoconjugate was comparable to that of doxorubicin alone.

Modulation of the platelet serotonin transporter by thermal balneotherapy: a study in healthy subjects.

PubMed

Baroni, S; Marazziti, D; Consoli, G; Picchetti, M; Catena-Dell'Osso, M; Galassi, A

2012-05-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 30 healthy volunteers before (t0) and 30 minutes after (t1) thermal balneotherapy with ozonized water, as compared with a similar group who underwent a bath in non-mineral water. MATERIALS AN METHODS: The SERT was evaluated by means of the specific binding of 3H-paroxetine (3H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of 3H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

Spectrophotometric analysis of flavonoid-DNA binding interactions at physiological conditions

NASA Astrophysics Data System (ADS)

Janjua, Naveed Kausar; Siddiqa, Asima; Yaqub, Azra; Sabahat, Sana; Qureshi, Rumana; Haque, Sayed ul

2009-12-01

Mode of interactions of three flavonoids [morin (M), quercetin (Q), and rutin (R)] with chicken blood ds.DNA (ck.DNA) has been investigated spectrophotometrically at different temperatures including body temperature (310 K) and at two physiological pH values, i.e. 7.4 (human blood pH) and 4.7 (stomach pH). The binding constants, Kf, evaluated using Benesi-Hildebrand equation showed that the flavonoids bind effectively through intercalation at both pH values and body temperature. Quercetin, somehow, showed greater binding capabilities with DNA. The free energies of flavonoid-DNA complexes indicated the spontaneity of their binding. The order of binding constants of three flavonoids at both pH values were found to be Kf(Q) > Kf(R) > Kf(M) and at 310 K.

Cobalt porphyrin-mediated oxygen transport in a polymer membrane. Effect of the cobalt porphyrin structure on the oxygen-binding reaction, oxygen-diffusion constants, and oxygen-transport efficiency

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

Nishide, Hiroyuki; Suzuki, Takayuki; Kawakami, Hiroyoshi

1994-05-12

New derivatives of (meso-[alpha],[alpha],[alpha],[alpha]-tetrakis(o-pivalamidophenyl)porphinato)cobalt (CoPs) were characterized by oxygen-binding equilibrium and rate constants of the cobalt centered in the porphyrins. They depended on the structure of the porphyrin; for example, the rate constants of oxygen binding and dissociation (k[sub on] and k[sub off]) for [alpha][sup 3][beta]-CoP[sub 4]P were 3 and 20 times as large as those for [alpha][sup 4]-CoB[sub 4]P, respectively. Oxygen transport through the polymer membranes containing CoPs as the fixed oxygen carriers was facilitated and was affected by the oxygen-binding character or the structure of CoPs. The logarithmically linear correlation of the oxygen-dissociation rate constant of CoPs (k[submore » off] = (3-66) x 10[sup 3] S[sup [minus]1]) with the diffusion constant of oxygen via CoPs fixed in the membranes (D[sub cc] = (3-140) x 10[sup [minus]9] cm[sup 2] s[sup [minus]1]) was given for those six CoP derivatives. 26 refs., 5 figs., 2 tabs.« less

Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

NASA Astrophysics Data System (ADS)

Lin, Hong; Kitova, Elena N.; Klassen, John S.

2014-01-01

Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

Chiral halogenated Schiff base compounds: green synthesis, anticancer activity and DNA-binding study

NASA Astrophysics Data System (ADS)

Ariyaeifar, Mahnaz; Amiri Rudbari, Hadi; Sahihi, Mehdi; Kazemi, Zahra; Kajani, Abolghasem Abbasi; Zali-Boeini, Hassan; Kordestani, Nazanin; Bruno, Giuseppe; Gharaghani, Sajjad

2018-06-01

Eight enantiomerically pure halogenated Schiff base compounds were synthesized by reaction of halogenated salicylaldehydes with 3-Amino-1,2-propanediol (R or S) in water as green solvent at ambient temperature. All compounds were characterized by elemental analyses, NMR (1H and 13C), circular dichroism (CD) and FT-IR spectroscopy. FS-DNA binding studies of these compounds carried out by fluorescence quenching and UV-vis spectroscopy. The obtained results revealed that the ligands bind to DNA as: (Rsbnd ClBr) > (Rsbnd Cl2) > (Rsbnd Br2) > (Rsbnd I2) and (Ssbnd ClBr) > (Ssbnd Cl2) > (Ssbnd Br2) > (Ssbnd I2), indicating the effect of halogen on binding constant. In addition, DNA-binding constant of the Ssbnd and R-enantiomers are different from each other. The ligands can form halogen bonds with DNA that were confirmed by molecular docking. This method was also measured the bond distances and bond angles. The study of obtained data can have concluded that binding affinity of the ligands to DNA depends on strength of halogen bonds. The potential anticancer activity of ligands were also evaluated on MCF-7 and HeLa cancer cell lines by using MTT assay. The results showed that the anticancer activity and FS-DNA interaction is significantly dependent on the stereoisomers of Schiff base compounds as R-enantiomers displayed significantly higher activity than S-enantiomers. The molecular docking was also used to illustrate the specific DNA-binding of synthesized compounds and groove binding mode of DNA interaction was proposed for them. In addition, molecular docking results indicated that there are three types of bonds (Hsbnd and X-bond and hX-bond) between synthesized compounds and base pairs of DNA.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

PubMed Central

Land, B R; Harris, W V; Salpeter, E E; Salpeter, M M

1984-01-01

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel. PMID:6584895

Binding behaviors of greenly synthesized silver nanoparticles - Lysozyme interaction: Spectroscopic approach

NASA Astrophysics Data System (ADS)

Roy, Swarup

2018-02-01

Interaction of greenly synthesized silver nanoparticles (SNP) and lysozyme (Lys) has been studied using spectroscopy. From UV-Vis study it is observed that a moderate association constant (Kapp) of 5.36 × 104 L/mol giving an indication of interaction. Fluorescence emission and time resolved study, confirm static mode of quenching phenomena and the binding constant (Kb) was 25.12, 3.98 and 1.99 × 103 L/mol at 298, 305 and 312 K respectively and the number of binding sites (n) was found to be ∼1. Using temperature dependent fluorimetric data, thermodynamic parameters calculated (Enthalpy change, ΔH = -143.95 kJ/mol, Entropy change, ΔS = -400.32 J/mol/K, Gibbs free energy change, ΔG = -24.66 kJ/mol at 298 K) and resulting insight indicative of weak force (van der Walls interaction & H-bonding) as key feature for the Lys-SNP interaction. By following Förster's non-radiative energy transfer (FRET) theory, average binding distance (r = 3.05 nm) was calculated and observed that nonradiative type energy transfer between SNP and Lys. What is more, circular dichroism (CD) spectra indicates presence of SNP does not display substantial alteration in the secondary structure of Lys. Hence, this results may be very useful for the well thought of essential aspects of binding between the Lys and SNP.

Theoretical studies of alkyl radicals in the NaY and HY zeolites.

PubMed

Ghandi, Khashayar; Zahariev, Federico E; Wang, Yan Alexander

2005-08-18

Interplay of quantum mechanical calculations and experimental data on hyperfine coupling constants of ethyl radical in zeolites at several temperatures was engaged to study the geometries and binding energies and to predict the temperature dependence of hyperfine splitting of a series of alkyl radicals in zeolites for the first time. The main focus is on the hyperfine interaction of alkyl radicals in the NaY and HY zeolites. The hyperfine splitting for neutral free radicals and free radical cations is predicted for different zeolite environments. This information can be used to establish the nature of the muoniated alkyl radicals in the NaY and HY zeolites via muSR experiments. The muon hyperfine coupling constants of the ethane radical cation in these zeolites are very large with relatively little dependence on temperature. It was found that the intramolecular dynamics of alkyl free radicals are only weakly affected by their strong binding to zeolites. In contrast, the substrate binding has a significant effect on their intermolecular dynamics.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

PubMed

Gervais, A; Lane, L K; Anner, B M; Lindenmayer, G E; Schwartz, A

1977-01-01

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

PubMed Central

Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

2012-01-01

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

Evaluation of DNA, BSA binding, and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline.

PubMed

Moradi, Zohreh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam

2018-02-01

In order to evaluate biological potential of a novel synthesized complex [Nd(dmp) 2 Cl 3 .OH 2 ] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (K b ) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (K b  = 2.7 ± 0.07 × 10 5 ) and fluorescence spectroscopy (K b  = 1.13 ± 0.03 × 10 5 ). The Stern-Volmer constant (K SV ), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

PubMed

Bertsch, M; Mayburd, A L; Kassner, R J

2003-02-15

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

STUDIES OF VERAPAMIL BINDING TO HUMAN SERUM ALBUMIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Chen, Sike; Hage, David S.

2008-01-01

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body. PMID:18980867

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed Central

Pinkney, M; Hoggett, J G

1988-01-01

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed

Pinkney, M; Hoggett, J G

1988-03-15

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

Structural changes in calcium-binding allergens: use of circular dichroism to study binding characteristics.

PubMed

Hebenstreit, D; Ferreira, F

2005-09-01

Several studies showed that calcium-binding proteins have a fixed place in the spectrum of allergenic substances. Often the binding of a calcium ion induces conformational changes and affects immunoglobulin E-binding to the allergen. Hence, the quantitative characterization of the binding to calcium is of importance to understand both the biologic and allergenic activity of these proteins. In the present study we describe a procedure for determining the stoichiometry and dissociation constant (K(D)) of calcium-binding allergens using circular dichroism (CD) techniques. For the experiments, we used recombinant Bet v 4, a two EF-hand allergen from birch pollen. Solutions of Bet v 4 were titrated with calcium and the change in molar ellipticity at 222 nm was monitored with a CD spectropolarimeter. The determination of the binding stoichiometry as well as of the K(D) for one EF-hand (4 microM) demonstrated the applicability of the method. CD-monitored calcium-titration of protein solutions represents a fast and easy method for determining the binding characteristics of calcium-binding allergens.

Electrochemical studies of DNA interaction and antimicrobial activities of MnII, FeIII, CoII and NiII Schiff base tetraazamacrocyclic complexes

NASA Astrophysics Data System (ADS)

Kumar, Anuj; Vashistha, Vinod Kumar; Tevatia, Prashant; Singh, Randhir

2017-04-01

Tetraazamacrocyclic complexes of MnII, FeIII, CoII and NiII have been synthesized by template method. These tetraazamacrocycles have been analyzed with various techniques like molar conductance, IR, UV-vis, mass spectral and cyclic voltammetric studies. On the basis of all these studies, octahedral geometry has been assigned to these tetraazamacrocyclic complexes. The DNA binding properties of these macrocyclic complexes have been investigated by electronic absorption spectra, fluorescence spectra, cyclic voltammetric and differential pulse voltammetric studies. The cyclic voltammetric data showed that ipc and ipa were effectively decreased in the presence of calf thymus DNA, which is a strong evidence for the interaction of these macrocyclic complexes with the calf thymus DNA (ct-DNA). The heterogeneous electron transfer rate constant found in the order: KCoII > KNiII > KMnII which indicates that CoII macrocyclic complex has formed a strong intercalated intermediate. The Stern-Volmer quenching constant (KSV) and voltammetric binding constant were found in the order KSV(CoII) > KSV(NiII) > KSV(MnII) and K+(CoII) > K+(NiII) > K+(MnII) which shows that CoII macrocyclic complex exhibits the high interaction affinity towards ct-DNA by the intercalation binding. Biological studies of the macrocyclic complexes compared with the standard drug like Gentamycin, have shown antibacterial activities against E. coli, P. aeruginosa, B. cereus, S. aureus and antifungal activity against C. albicans.

Theoretical study of metal noble-gas positive ions

NASA Technical Reports Server (NTRS)

Bauschlicher, Charles W., Jr.; Partridge, Harry; Langhoff, Stephen R.

1989-01-01

Theoretical calculations have been performed to determine the spectroscopic constant for the ground and selected low-lying electronic states of the transition-metal noble-gas ions Var(+), FeAr(+), CoAr(+), CuHe(+), CuAr(+), and CuKr(+). Analogous calculations have been performed for the ground states of the alkali noble-gas ions LiAr(+), LiKr(+), NaAr(+), and KAr(+) and the alkaline-earth noble-gas ion MgAr(+) to contrast the difference in binding energies between the simple and transition-metal noble-gas ions. The binding energies increase with increasing polarizability of the noble-gas ions, as expected for a charge-induced dipole bonding mechanism. It is found that the spectroscopic constants of the X 1Sigma(+) states of the alkali noble-gas ions are well described at the self-consistent field level. In contrast, the binding energies of the transition-metal noble-gas ions are substantially increased by electron correlation.

Coarse-graining, Electrostatics and pH effects in phospholipid systems

NASA Astrophysics Data System (ADS)

Travesset, Alex; Vangaveti, Sweta

2010-03-01

We introduce a minimal free energy describing the interaction of charged groups and counterions including both classical electrostatic and specific interactions. The predictions of the model are compared against the standard model for describing ions next to charged interfaces, consisting of Poisson-Boltzmann theory with additional constants describing ion binding, which are specific to the counterion and the interfacial charge (``chemical binding''). It is shown that the ``chemical'' model can be appropriately described by an underlying ``physical'' model over several decades in concentration, but the extracted binding constants are not uniquely defined, as they differ depending on the particular observable quantity being studied. It is also shown that electrostatic correlations for divalent (or higher valence) ions enhance the surface charge by increasing deprotonation, an effect not properly accounted within chemical models. The model is applied to the charged phospholipids phosphatidylserine, Phosphatidc acid and Phosphoinositides and implications for different biological processes are discussed.

Fluorescent studies on the interaction of DNA and ternary lanthanide complexes with cinnamic acid-phenanthroline and antibacterial activities testing.

PubMed

Sun, Hui-Juan; Wang, Ai-Ling; Chu, Hai-Bin; Zhao, Yong-Liang

2015-03-01

Twelve lanthanide complexes with cinnamate (cin(-) ) and 1,10-phenanthroline (phen) were synthesized and characterized. Their compositions were assumed to be RE(cin)3 phen (RE(3+)  = La(3+) , Pr(3+) , Nd(3+) , Sm(3+) , Eu(3+) , Gd(3+) , Tb(3+) , Dy(3+) , Ho(3+) , Tm(3+) , Yb(3+) , Lu(3+) ). The interaction mode between the complexes and DNA was investigated by fluorescence quenching experiment. The results indicated the complexes could bind to DNA and the main binding mode is intercalative binding. The fluorescence quenching constants of the complexes increased from La(cin)3 phen to Lu(cin)3 phen. Additionally, the antibacterial activity testing showed that the complexes exhibited excellent antibacterial ability against Escherichia coli, and the changes of antibacterial ability are in agreement with that of the fluorescence quenching constants. Copyright © 2014 John Wiley & Sons, Ltd.

Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH

PubMed Central

Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun

2012-01-01

The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant. PMID:22464680

The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin

PubMed Central

Hanner, Markus; Schmalhofer, William A.; Munujos, Petraki; Knaus, Hans-Günther; Kaczorowski, Gregory J.; Garcia, Maria L.

1997-01-01

Transient expression of either α or α+β subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the β subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged 125I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. PMID:9096310

Interaction of antitumor drug Sn(CH 3) 2Cl 2 with DNA and RNA

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Sobhanmanesh, Amir; Esm-Hosseini, Majid; Alimoghaddam, Kamran; Tajmir-Riahi, Heidar Ali

2005-08-01

Sn(CH3)2Cl2 exerts its antitumor activity in a specific way. Unlike anticancer cis-Pt(NH3)2Cl2 drug which binds strongly to the nitrogen atoms of DNA bases, Sn(CH3)2Cl2 shows no major affinity towards base binding. Thus, the mechanism of action by which tinorganometallic compounds exert antitumor activity would be different from that of the cisplatin drug. The aim of this study was to examine the binding of Sn(CH3)2Cl2 with calf thymus DNA and yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentrations of DNA and RNA and various molar ratios of Sn(CH3)2Cl2/DNA (phosphate) and Sn(CH3)2Cl2/RNA of 1/40, 1/20, 1/10, 1/5. Fourier transform infrared (FTIR) and UV-visible difference spectroscopic methods were used to determine the Sn(CH3)2Cl2 binding mode, binding constant, sequence selectivity and structural variations of Sn(CH3)2Cl2/DNA and Sn(CH3)2Cl2/RNA complexes in aqueous solution. Sn(CH3)2Cl2 hydrolyzes in water to give Sn(CH3)2(OH)2 and [Sn(CH3)2(OH)(H2O)n]+ species. Spectroscopic evidence showed that interaction occurred mainly through (CH3)2Sn(IV) hydroxide and polynucleotide backbone phosphate group with overall binding constant of K(Sn(CH3)2Cl2-DNA)=1.47×105 M-1 and K(Sn(CH3)2Cl2-RNA)=7.33×105 M-1. Sn(CH3)2Cl2 induced no biopolymer conformational changes with DNA remaining in the B-family structure and RNA in A-conformation upon drug complexation.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Solvent and substituent effects on aggregation constants of perylene bisimide π-stacks--a linear free energy relationship analysis.

PubMed

Chen, Zhijian; Fimmel, Benjamin; Würthner, Frank

2012-08-14

A series of six perylene bisimides (PBIs) with hydrophilic and hydrophobic side chains at the imide nitrogens were applied for a comparative study of the solvent and structural effects on the aggregation behaviour of this class of dyes. A comparison of the binding constants in tetrachloromethane at room temperature revealed the highest binding constant of about 10(5) M(-1) for a PBI bearing 3,4,5-tridodecyloxyphenyl substituents at the imide nitrogens, followed by 3,4,5-tridodecylphenyl and alkyl-substituted PBIs, whereas no aggregation could be observed in the accessible concentration range for PBIs equipped with bulky 2,6-diisopropylphenyl substituents at the imide nitrogens. The aggregation behaviour of three properly soluble compounds was investigated in 17 different solvents covering a broad polarity range from nonpolar n-hexane to highly polar DMSO and water. Linear free energy relationships (LFER) revealed a biphasic behaviour between Gibbs free energies of aggregation and common empirical solvent polarity scales indicating particularly strong π-π stacking interactions in nonpolar aliphatic and polar alcoholic solvents whilst the weakest binding is observed in dichloromethane and chloroform. Accordingly, PBI aggregation is dominated by electrostatic interactions in nonpolar solvents and by solvophobic interactions in protic solvents. In water, the aggregation constant is increased far beyond LFER expectations pointing at a pronounced hydrophobic effect.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

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

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

Biophysical influence of coumarin 35 on bovine serum albumin: Spectroscopic study

NASA Astrophysics Data System (ADS)

Bayraktutan, Tuğba; Onganer, Yavuz

2017-01-01

The binding mechanism and protein-fluorescence probe interactions between bovine serum albumin (BSA) and coumarin 35 (C35) was investigated by using UV-Vis absorption and fluorescence spectroscopies since they remain major research topics in biophysics. The spectroscopic data indicated that a fluorescence quenching process for BSA-C35 system was occurred. The fluorescence quenching processes were analyzed using Stern-Volmer method. In this regard, Stern-Volmer quenching constants (KSV) and binding constants were calculated at different temperatures. The distance r between BSA (donor) and C35 (acceptor) was determined by exploiting fluorescence resonance energy transfer (FRET) method. Synchronous fluorescence spectra were also studied to observe information about conformational changes. Moreover, thermodynamics parameters were calculated for better understanding of interactions and conformational changes of the system.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

PubMed Central

Gleitsman, Kristin R.

2014-01-01

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight.

PubMed

Shamsi, Anas; Ahmed, Azaj; Bano, Bilqees

2018-05-01

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10 4  M -1 implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.

Modeling of the solution interaction properties of plastic materials used in pharmaceutical product container systems.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy; Sadain, Salma

2009-01-01

Material/water equilibrium binding constants (Eb) were determined for 14 organic solutes and 17 plastic raw materials that could be used in pharmaceutical product container systems. Correlations between the measured binding constants and the organic solute's octanol/water and hexane/water partition coefficients were obtained. In general, while the materials examined exhibited a wide range of binding characteristics, the tested materials by and large fell within two broad classes: (1) those that were octanol-like in their binding characteristics, and (2) those that were hexane-like. Materials of the same class (e.g., polypropylenes) generally had binding models that were very similar. Rank ordering of the materials in terms of their magnitude of drug binding (least binding to most binding) was as follows: polypropylene < polyethylene < polyamide < styrene-ethylene-butylene-styrene < copolyester ether elastomer approximately equal to amine-terminated poly fatty acid amide polymer. The utilization of the developed models to estimate drug loss via sorption by the container is discussed.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.

PubMed

Wang, Qin; Sheng, Xin; Horner, John H; Newcomb, Martin

2009-08-05

Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially nonexistent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers. Pseudo-first-order rate constants obtained at -50 degrees C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (K(bind)) and first-order rate constants for the oxidation reactions (k(ox)). Representative results are K(bind) = 214 M(-1) and k(ox) = 0.48 s(-1) for oxidation of benzyl alcohol. For the dideuterated substrate C(6)H(5)CD(2)OH, kinetics were studied between -50 and -25 degrees C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at -50 degrees C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 degrees C of both the rate constant for oxidation of C(6)H(5)CD(2)OH and the KIE for the nondeuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at -50 degrees C.

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.

Spectroscopic and molecular modeling studies of the interaction between cytidine and human serum albumin and its analytical application.

PubMed

Cui, Fengling; Wang, Junli; Yao, Xiaojun; Wang, Li; Zhang, Qiangzhai; Qu, Guirong

In this study, the interaction between cytidine and human serum albumin (HSA) was investigated for the first time by fluorescence spectroscopy in combination with UV absorption spectrum and molecular modeling under simulative physiological conditions. Experimental results indicated that cytidine had a strong ability to quench the intrinsic fluorescence of human serum albumin. The binding constants (K) at different temperatures, thermodynamic parameter enthalpy changes (DeltaH) and entropy changes (DeltaS) of HSA-cytidine had been calculated according to the relevant fluorescence data, which indicated that the hydrophobic and electrostatic interactions played a major role, which was in agreement with the results of molecular modeling study. In addition, the effects of other ions on the binding constants were also studied. Furthermore, synchronous fluorescence technology was successfully applied to the determination of human serum albumin added into the cytidine solution.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

PubMed

Boubeta, Fernando M; Bieza, Silvina A; Bringas, Mauro; Estrin, Darío A; Boechi, Leonardo; Bari, Sara E

2018-06-19

The reaction of hydrogen sulfide (H 2 S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-Fe III ) and myoglobin (Mb-Fe III ), from the estimation of the intrinsic binding constants of the species H 2 S and hydrosulfide (HS - ), and the computational description of the overall binding process. Our results show that H 2 S and HS - are the main reactive species in Mb-Fe III and MP11-Fe III , respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS - to Mb-Fe III was negligible, the binding of H 2 S to MP11-Fe III was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H 2 S to Mb-Fe III and that the binding of HS - is prevented in the access to the protein matrix. For the MP11-Fe III case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H 2 S and HS - are capable of binding a ferric heme moiety.

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

PubMed

Wang, J; Velotta, J B; McDonough, A A; Farley, R A

2001-10-01

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding.

PubMed

Acharya, Bipul R; Bhattacharyya, Bhabatarak; Chakrabarti, Gopal

2008-07-29

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. The accuracy of binding free energy calculations based on molecular dynamics simulations.

PubMed

Hou, Tingjun; Wang, Junmei; Li, Youyong; Wang, Wei

2011-01-24

The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) and the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods calculate binding free energies for macromolecules by combining molecular mechanics calculations and continuum solvation models. To systematically evaluate the performance of these methods, we report here an extensive study of 59 ligands interacting with six different proteins. First, we explored the effects of the length of the molecular dynamics (MD) simulation, ranging from 400 to 4800 ps, and the solute dielectric constant (1, 2, or 4) on the binding free energies predicted by MM/PBSA. The following three important conclusions could be observed: (1) MD simulation length has an obvious impact on the predictions, and longer MD simulation is not always necessary to achieve better predictions. (2) The predictions are quite sensitive to the solute dielectric constant, and this parameter should be carefully determined according to the characteristics of the protein/ligand binding interface. (3) Conformational entropy often show large fluctuations in MD trajectories, and a large number of snapshots are necessary to achieve stable predictions. Next, we evaluated the accuracy of the binding free energies calculated by three Generalized Born (GB) models. We found that the GB model developed by Onufriev and Case was the most successful model in ranking the binding affinities of the studied inhibitors. Finally, we evaluated the performance of MM/GBSA and MM/PBSA in predicting binding free energies. Our results showed that MM/PBSA performed better in calculating absolute, but not necessarily relative, binding free energies than MM/GBSA. Considering its computational efficiency, MM/GBSA can serve as a powerful tool in drug design, where correct ranking of inhibitors is often emphasized.

Unraveling the binding interaction of a bioactive pyrazole-based probe with serum proteins: Relative concentration dependent 1:1 and 2:1 probe-protein stoichiometries.

PubMed

Kundu, Pronab; Chattopadhyay, Nitin

2018-06-15

Molecular interactions and binding of probes/drugs with biomacromolecular systems are of fundamental importance in understanding the mechanism of action and hence designing of proactive drugs. In the present study, binding interactions of a biologically potent fluorophore, (E)-1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (DSDP) with two serum transport proteins, human serum albumin and bovine serum albumin, have been investigated exploiting multi-spectroscopic techniques. The spectrophotometric and fluorometric studies together with fluorescence quenching, fluorescence anisotropy, urea induced denaturation studies and fluorescence lifetime measurements reveal strong binding of DSDP with both the plasma proteins. Going beyond the vast literature data mostly providing 1:1 probe-protein complexation, the present investigation portrays 2:1 probe-protein complex formation at higher relative probe concentration. A newer approach has been developed to have an estimate of the binding constants varying the concentration of the protein, instead of the usual practice of varying the probe. The binding constants for the 2:1 DSDP-protein complexes are determined to be 1.37 × 10 10  M -2 and 1.47 × 10 10  M -2 for HSA and BSA respectively, while those for the 1:1 complexation process come out to be 1.85 × 10 5  M -1 and 1.73 × 10 5  M -1 for DSDP-HSA and DSDP-BSA systems respectively. Thermodynamic analysis at different temperatures implies that the forces primarily involved in the binding process are hydrogen bonding and hydrophobic interactions. Competitive replacement studies with known site markers and molecular docking simulations direct to the possible locations and binding energies of DSDP with the two serum proteins, corroborating well with the experimental results. Copyright © 2018 Elsevier B.V. All rights reserved.

Interaction of Sulforaphane with DNA and RNA

PubMed Central

Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

2015-01-01

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p: reinterpretation of recent single molecule experiments.

PubMed

Stigter, Dirk

2004-07-01

Brewer et al. (Biophys. J. 85 (2003) 2519-2524) have studied the compaction of dsDNA in a double flow cell by observing the extension of stained DNA tethered in buffer solutions with or without Abf2p. They use a Langmuir adsorption model in which one Abf2p molecule adsorbs on one site on the DNA, and the binding constant, K, is given as the ratio of the experimental rates of adsorption and desorption. This paper presents an improved interpretation. Instead of Langmuir adsorption we use the more appropriate McGhee-von Hippel (J. Mol. Biol. 86 (1974) 469-489) theory for the adsorption of large ligands to a one-dimensional lattice. We assume that each adsorbed molecule shortens the effective contour length of DNA by the foot print of Abf2p of 27 base pairs. When Abf2p adsorbs to DNA stretched in the flowing buffer solution, we account for a tension effect that decreases the adsorption rate and the binding constant by a factor of 2 to 4. The data suggest that the accessibility to Abf2p decreases significantly with increasing compaction of DNA, resulting in a lower adsorption rate and a lower binding constant. The kinetics reported by Brewer et al. (Biophys. J. 85 (2003) 2519-2524) lead to a binding constant K=3.6 x 10(6) M(-1) at the beginning, and to K=5 x 10(5) M(-1) near the end of a compaction run, more than an order of magnitude lower than the value K=2.57 x 10(7) M(-1) calculated by Brewer et al. (Biophys. J. 85 (2003) 2519-2524).

Binding of vitamin A with milk α- and β-caseins.

PubMed

Bourassa, P; N'soukpoé-Kossi, C N; Tajmir-Riahi, H A

2013-05-01

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(retinol-)(α)(-caseins)=1.21 (±0.4)×10(5) M(-1) and K(retinol-)(β)(-caseins)=1.11 (±0.5)×10(5) M(-1) and K(retinoic acid-)(α)(-caseins)=6.2 (±0.6)×10(4) M(-1) and K(retinoic acid-)(β)(-caseins)=6.3 (±0.6)×10(4) M(-1). The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid-casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

Locating the binding sites of folic acid with milk α- and β-caseins.

PubMed

Bourassa, P; Tajmir-Riahi, H A

2012-01-12

We located the binding sites of folic acid with milk α- and β-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-α-caseins) = 4.8 (±0.6) × 10(4) M(-1) and K(folic acid-β-caseins) = 7.0 (±0.9) × 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (±0.4) for α-casein and 1.4 (±0.3) for β-casein complexes. Molecular modeling showed different binding sites for folic acid on α- and β-caseins. The participation of several amino acids in folic acid-protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-α-casein) and -8.1 kcal/mol (acid-β-casein). Folic acid complexation altered protein secondary structure by the reduction of α-helix from 35% (free α-casein) to 33% (acid-complex) and 32% (free β-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.

Interactions and diffusion in fine-stranded β-lactoglobulin gels determined via FRAP and binding.

PubMed

Schuster, Erich; Hermansson, Anne-Marie; Ohgren, Camilla; Rudemo, Mats; Lorén, Niklas

2014-01-07

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na2-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na2-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

PubMed Central

Zubrienė, Asta; Matulienė, Jurgita; Baranauskienė, Lina; Jachno, Jelena; Torresan, Jolanta; Michailovienė, Vilma; Cimmperman, Piotras; Matulis, Daumantas

2009-01-01

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding. PMID:19582223

Contacts between the factor TUF and RPG sequences.

PubMed

Vignais, M L; Huet, J; Buhler, J M; Sentenac, A

1990-08-25

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

Nanobiological studies on drug design using molecular mechanic method.

PubMed

Ghaheh, Hooria Seyedhosseini; Mousavi, Maryam; Araghi, Mahmood; Rasoolzadeh, Reza; Hosseini, Zahra

2015-01-01

Influenza H1N1 is very important worldwide and point mutations that occur in the virus gene are a threat for the World Health Organization (WHO) and druggists, since they could make this virus resistant to the existing antibiotics. Influenza epidemics cause severe respiratory illness in 30 to 50 million people and kill 250,000 to 500,000 people worldwide every year. Nowadays, drug design is not done through trial and error because of its cost and waste of time; therefore bioinformatics studies is essential for designing drugs. This paper, infolds a study on binding site of Neuraminidase (NA) enzyme, (that is very important in drug design) in 310K temperature and different dielectrics, for the best drug design. Information of NA enzyme was extracted from Protein Data Bank (PDB) and National Center for Biotechnology Information (NCBI) websites. The new sequences of N1 were downloaded from the NCBI influenza virus sequence database. Drug binding sites were assimilated and homologized modeling using Argus lab 4.0, HyperChem 6.0 and Chem. D3 softwares. Their stability was assessed in different dielectrics and temperatures. Measurements of potential energy (Kcal/mol) of binding sites of NA in different dielectrics and 310K temperature revealed that at time step size = 0 pSec drug binding sites have maximum energy level and at time step size = 100 pSec have maximum stability and minimum energy. Drug binding sites are more dependent on dielectric constants rather than on temperature and the optimum dielectric constant is 39/78.

Interaction of a copper (II) complex containing an artificial sweetener (aspartame) with calf thymus DNA.

PubMed

Shahabadi, Nahid; Khodaei, Mohammad Mehdi; Kashanian, Soheila; Kheirdoosh, Fahimeh

2014-01-01

A copper (II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl2⋅2H2O, was synthesized and characterized. In vitro binding interaction of this complex with native calf thymus DNA (CT-DNA) was studied at physiological pH. The interaction was studied using different methods: spectrophotometric, spectrofluorometric, competition experiment, circular dichroism (CD) and viscosimetric techniques. Hyperchromicity was observed in UV absorption band of Cu(APM)2Cl2⋅2H2O. A strong fluorescence quenching reaction of DNA to Cu(APM)2Cl2⋅2H2O was observed and the binding constants (Kf) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be+89.3 kJ mol(-1) and+379.3 J mol(-1) K(-1) according to Van't Hoff equation which indicated that reaction is predominantly entropically driven. Experimental results from spectroscopic methods were comparable and further supported by viscosity measurements. We suggest that Cu(APM)2Cl2⋅2H2O interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 8×10+4 M(-1). Binding of this copper complex to DNA was found to be stronger compared to aspartame which was studied recently. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction studies of resistomycin from Streptomyces aurantiacus AAA5 with calf thymus DNA and bovine serum albumin

NASA Astrophysics Data System (ADS)

Vijayabharathi, R.; Sathyadevi, P.; Krishnamoorthy, P.; Senthilraja, D.; Brunthadevi, P.; Sathyabama, S.; Priyadarisini, V. Brindha

2012-04-01

Resistomycin, a secondary metabolite produced by Streptomyces aurantiacus AAA5. The binding interaction of resistomycin with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorimetry, circular dichroism (CD) and synchronous fluorescence techniques under physiological conditions in vitro. Absorption spectral studies along with the fluorescence competition with ethidium bromide measurements and circular dichroism clearly suggest that the resistomycin bind with CT DNA relatively strong via groove binding. BSA interaction results revealed that the drug was found to quench the fluorescence intensity of the protein through a static quenching mechanism. The number of binding sites 'n' and apparent binding constant 'K' calculated according to the Scatchard equation exhibit a good binding property to bovine serum albumin protein. In addition, the results observed from synchronous fluorescence measurements clearly demonstrate the occurrence of conformational changes of BSA upon addition of the test compound.

Hydrazinylpyridine based highly selective optical sensor for aqueous source of carbonate ions: Electrochemical and DFT studies

NASA Astrophysics Data System (ADS)

Thimaradka, Vikram; Pangannaya, Srikala; Mohan, Makesh; Trivedi, Darshak R.

2018-03-01

A series of new receptors PDZ1-3 based on 2-(arylidenehydrazinyl)pyridines have been designed and synthesized for the detection of biologically and environmentally important ions. The colorimetric detection of CO32 - using neutral organic receptor PDZ-1 has been achieved with characteristic visual colour change from yellow to green accompanied by a large redshift of 215 nm in absorption maxima. UV-Vis spectroscopic and cyclic voltammetric studies reveal the stoichiometry of binding and electrochemistry of host-guest complex formation. The binding constant was found to be 0.77 × 104 M- 2. In addition, electrochemical studies provide an insight into the stability of the complex. DFT studies performed on the PDZ-1 and PDZ-1 - CO32 - complex reveal the binding mechanism involved in the anion detection process. PDZ-1 is highly selective for carbonate and does not show any colorimetric response towards any other anions or cations, while PDZ-2 and PDZ-3 remain inactive in the ion detection process. The limit of detection (LOD) and limit of quantification (LOQ) of PDZ-1 for carbonate was found to be 0.11 mM and 0.36 mM respectively. Considerable binding constant and limit of detection make PDZ-1 to be used as a real time sensor for the detection of carbonate in environmental and biological samples.

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

PubMed

Simões, Inês C M; Costa, Inês P D; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

2017-01-23

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔG binding ) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔG binding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol -1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol -1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

A Thermodynamic Description of the Adsorption of Simple Water-Soluble Peptoids to Silica.

PubMed

Calkins, Anna L; Yin, Jennifer; Rangel, Jacenda L; Landry, Madeleine R; Fuller, Amelia A; Stokes, Grace Y

2016-11-08

The first report of a water-soluble peptoid adsorbed to silica monitored by second harmonic generation (SHG) at the liquid/solid interface is presented here. The molecular insights gained from these studies will inform the design and preparation of novel peptoid coatings. Simple 6- and 15-residue peptoids were dissolved in phosphate buffered saline and adsorbed to bare silica surfaces. Equilibrium binding constants and relative surface concentrations of adsorbed peptoids were determined from fits to the Langmuir model. Complementary fluorescence spectroscopy studies were used to quantify the maximum surface excess. Binding constants, determined here by SHG, were comparable to those previously reported for cationic proteins and small molecules. Enthalpies and free energies of adsorption were determined to elucidate thermodynamic driving forces. Circular dichroism spectra confirm that minimal conformational changes occur when peptoids are adsorbed to silica while pH studies indicate that electrostatic interactions impact adsorption.

Effect of halo-substituted aromatic salts on counterion binding constants obtained from cationic nanoparticle catalyzed reactions of piperidine and phenyl salicylate

NASA Astrophysics Data System (ADS)

Fagge, Ibrahim I.; Yusof, Nor Saadah M.; Zain, Sharifuddin Md; Khan, M. Niyaz

2017-12-01

Halo-substitutions at 3-position of benzene ring of the salts of aromatic carboxylate, MX, revealed the effect of two different halide ions (Br- and Cl-) on the counterion binding constants obtained from cationic nanoparticle catalyzed piperidinolysis of ionized phenyl salicylate (PhS-). The values of observed rate constant, kobs, determined at a constant total concentration of cetyltrimethylammonium bromide, [CTABr]T, piperidine, ([P]T), [PhS-]T, NaOH, and various concentration of MX (MX = 3-BrC6H4CO2Na and 3-ClC6H4CO2Na), were determined using UV-visible X spectrophotometric technique at 35 °C and 370 nm. The average value of nanoparticle binding constant, KXBr, for X- = 3-BrC6H4CO2- (RXBr = 57) was found to be about 2-fold larger than that for X- = 3-ClC6H4CO2- (RXBr = 30). These XX values were dependent of substituents 3-Br and 3-Cl, and independent of [CTABr]T. Both are related to the presence of different extent of viscoelastic worm-like nanoparticles formation in the [CTABr]T of 6 and 10 mM.

Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

PubMed

Qian, Cheng; Kovalchik, Kevin A; MacLennan, Matthew S; Huang, Xiaohua; Chen, David D Y

2017-06-01

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular recognition of curcumin (Indian Ayurvedic medicine) by the supramolecular probe, p-t-butyl calix(8)arene

NASA Astrophysics Data System (ADS)

Meenakshi, C.; Jayabal, P.; Ramakrishnan, V.

2014-06-01

The thermodynamic property of the host-guest complexes formed between the curcumin, component of Indian Ayurvedic medicine turmeric, a drug molecule, with the supra molecule, p-t-butyl calix(8)arene was studied. p-t-Butyl calix(8)arene has been used as a host molecule and curcumin as a guest molecule. Optical absorption spectral studies were carried out to investigate the molecular recognition properties of p-t-butyl calix(8)arene with curcumin. The stochiometry of the host-guest complexes formed and the binding constant were determined. An interesting 1:1 and 4:1 stochiometric host-guest complexes were formed. Job's continuous method of variation and Benesi-Hildebrand expression were used for the determination of binding constant and the stochiometry of the host-guest complex formed.

New phthalimide-appended Schiff bases: Studies of DNA binding, molecular docking and antioxidant activities.

PubMed

Nayab, Pattan Sirajuddin; Akrema; Ansari, Istikhar A; Shahid, Mohammad; Rahisuddin

2017-08-01

Herein, we investigated new phthalimide-based Schiff base molecules as promising DNA-binding and free radical scavenging agents. Physicochemical properties of these molecules were demonstrated on the basis of elemental analysis, ultraviolet-visible (UV-Vis), infra-red (IR), 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. All spectral data are agreed well with the proposed Schiff base framework. The DNA-binding potential of synthesized compounds were investigated by means of UV-visible, fluorescence, iodide quenching, circular dichroism, viscosity and thermal denaturation studies. The intrinsic binding constants (K b ) were calculated from absorption studies were found to be 1.1 × 10 4 and 1.0 × 10 4  M -1 for compounds 2a and 2b suggesting that compound 2a binding abilities with DNA were stronger than the compound 2b. Our studies showed that the presented compounds interact with DNA through groove binding. Molecular docking studies were carried out to predict the binding between Ct-DNA and test compounds. Interestingly, in silico predictions were corroborated with in vitro DNA-binding conclusions. Furthermore, the title compounds displayed remarkable antioxidant activity compared with reference standard. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

NASA Astrophysics Data System (ADS)

D'Aquino, J. Alejandro; Ringe, Dagmar

2006-08-01

The diphtheria toxin repressor, DtxR, is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear (1 - 3). Calorimetric techniques have demonstrated that while binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10-7, binding site 2 (primary) is a low affinity binding site with a binding constant of 6.3 × 10-4. These two binding sites act independently and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here and the previously reported DtxR(H79A) (4) has allowed us to propose a mechanism of metal ion activation for DtxR.

Interaction of thioflavin T with amyloid fibrils: stoichiometry and affinity of dye binding, absorption spectra of bound dye.

PubMed

Sulatskaya, Anna I; Kuznetsova, Irina M; Turoverov, Konstantin K

2011-10-06

The fluorescence of the benzothiazole dye thioflavin T (ThT) is a well-known test for amyloid fibril formation. It has now become evident that ThT can also be used for structural investigations of amyloid fibrils and even for the treatment of amyloid diseases. In this case, one of the most urgent problems is an accurate determination of ThT-amyloid fibril binding parameters: the number of binding modes, stoichiometry, and binding constant for each mode. To obtain information concerning the ThT-amyloid fibril binding parameters, we propose to use absorption spectrophotometry of solutions prepared by equilibrium microdialysis. This approach is inherently designed for the determination of dye-receptor binding parameters. However, it has been very rarely used in the study of dye-protein interactions and has never been used to study the binding parameters of ThT or its analogues to amyloid fibrils. We showed that, when done in corpore, this approach enables the determination of not only binding parameters but also the absorption spectrum and molar extinction coefficient of ThT bound to sites of different binding modes. The proposed approach was used for the examination of lysozyme amyloid fibrils. Two binding modes were found for the ThT-lysozyme amyloid fibril interaction. These binding modes have significantly different binding constants (K(b1) = 7.5 × 10(6) M(-1), K(b2) = 5.6 × 10(4) M(-1)) and a different number of dye binding sites on the amyloid fibrils per protein molecule (n(1) = 0.11, n(2) = 0.24). The absorption spectra of ThT bound to sites of different modes differ from each other (ε(b1,max) = 5.1 × 10(4) M(-1) cm(-1), ε(b2,max) = 6.7 × 10(4) M(-1)cm(-1), λ(max) = 449 nm) and significantly differ from that of free ThT in aqueous solution (ε(max) = 3.2 × 10(4) M(-1)cm(-1), λ(max) = 412 nm). © 2011 American Chemical Society

SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

NASA Astrophysics Data System (ADS)

Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

2016-04-01

Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

PubMed

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

2016-05-15

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Entrapment of Alpha1-Acid Glycoprotein in High-Performance Affinity Columns for Drug-Protein Binding Studies

PubMed Central

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S.

2015-01-01

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (± 0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. PMID:26627938

Metal binding stoichiometry and isotherm choice in biosorption

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

Schiewer, S.; Wong, M.H.

1999-11-01

Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lowermore » metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.« less

Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen.

PubMed

Esteves, Catarina V; Esteban-Gómez, David; Platas-Iglesias, Carlos; Tripier, Raphaël; Delgado, Rita

2018-05-11

The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid-base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi 3- , ATP 4- , ADP 3- , AMP 2- , PhPO 4 2- , and HPO 4 2- ). On the one hand, the acid-base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn 2 bmce(HPPi)] + . Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn 2 bmce] 4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.

Possibility of using Rhodamine B dye in diagnosis of some men's diseases

NASA Astrophysics Data System (ADS)

Khodjayev, Gayrat; Ismailov, Zafar F.; Kurtaliev, Eldar N.; Nizomov, Negmat; Khaydarova, Feruza U.; Hamidov, Zariddin; Khakimova, Dilorom P.

2007-09-01

The functional differences of human blood serum albumin in norm and at different patologic process were studied by spectral-luminescent method by comparison of binding constant (K) and concentration of binding sites (N) values of rhodamine B dye with blood serum. It was shown that K and N of rhodamine B dye with blood serum of sick men is decreased as compared to that for healthy men.

Yeast hexokinase. A fluorescence temperature-jump study of the kinetics of the binding of glucose to the monomer forms of hexokinases P-I and P-II.

PubMed

Hoggett, J G; Kellett, G L

1976-09-15

The binding of glucose to the monomeric forms of hexokinases P-I and P-II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol l-1 KCl has been studied using the fluorescence temperature-jump technique. For both isozymes only one relaxation time was observed; values of tau-1 increased linearly with increasing concentration of free reacting partners. The apparent second-order rate constant for association was about 2 X 10(6) 1 mol-1 s-1 for both isozymes; the differences in the stabilities of the complexes with P-I and P-II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P-I than P-II (approximately 300 s-1 and 1100 s-1 respectively). Although the kinetic data are compatible with a single-step mechanism for glucose binding the association rate constant was much lower than that expected for a diffusion-limited rate of encounter. Other mechanisms for describing an induced-fit are discussed. It is shown that the data are incompatible with a slow 'prior-isomerization' pathway of substrate binding, but are consistent with a 'substrate-guided' pathway involving isomerization of the enzyme-substrate complex.

Thermal balneotherapy induces changes of the platelet serotonin transporter in healthy subjects.

PubMed

Marazziti, Donatella; Baroni, Stefano; Giannaccini, Gino; Catena Dell'Osso, Mario; Consoli, Giorgio; Picchetti, Michela; Carlini, Marina; Massimetti, Gabriele; Provenzano, Serafina; Galassi, Antonio

2007-10-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 20 healthy volunteers before (t0) and 30 min after (t1) thermal balneotherapy with ozonized water of Montecatini spa, as compared with a similar group who underwent a bath in non-mineral water. The SERT was evaluated by means of the specific binding of (3)H-paroxetine ((3)H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of (3)H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

The effect of Cu 2+ or Fe 3+ on the noncovalent binding of rutin with bovine serum albumin by spectroscopic analysis

NASA Astrophysics Data System (ADS)

Li, Daojin; Zhu, Mei; Xu, Chen; Chen, Jianjun; Ji, Baoming

2011-01-01

The interaction of rutin to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence spectra and ultraviolet-visible (UV-vis) spectra at pH 7.40. There are also some metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction of drugs with proteins is crucial. Therefore, we have studied the effect of Cu 2+ or Fe 3+ on the interaction between rutin and BSA by using spectroscopic technique at pH 7.40, for the first time. The results of fluorescence titration indicated that rutin could quench the intrinsic fluorescence of BSA in a static quenching way. The binding sites number ( n), the binding constant ( K) and the spatial-distance ( r) of rutin with BSA without or with Cu 2+ or Fe 3+ at 310 K were calculated. The result showed that the presence of Cu 2+ or Fe 3+ increased the binding constant and changed the binding distance between the acceptor and the donor, which possibly results from the formation of metal ions-BSA complex. The effect of rutin on the conformation of BSA was also analyzed using UV under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that rutin is situated within subdomain IIA of BSA.

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.

Thermodynamic and Kinetics Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

PubMed Central

Wafer, Lucas N.; Streicher, Werner W.; McCallum, Scott A.; Makhatadze, George I.

2012-01-01

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone 1H and 15N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic Kon rate constants observed in this study, Kon ≥1×107 M-1s-1, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets. PMID:22913742

The investigation of the interaction between piracetam and bovine serum albumin by spectroscopic methods

NASA Astrophysics Data System (ADS)

Guo, Xingjia; Han, Xiaowei; Tong, Jian; Guo, Chuang; Yang, Wenfeng; Zhu, Jifen; Fu, Bing

2010-03-01

The interaction between piracetam (OPA) with bovine serum albumin (BSA) has been thoroughly studied by fluorescence quenching technique in combination with UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichroism (CD) spectroscopies under the simulative physiological conditions. The quenching of BSA fluorescence by OPA was found to be a static quenching process. The binding constants ( K a) are 3.014, 2.926, and 2.503 × 10 3 M -1 at 292, 298, and 309 K, respectively. According to the van't Hoff equation, the thermodynamic functions standard enthalpy (Δ H) and standard entropy (Δ S) for the reaction were calculated to be -74.560 kJ mol -1 and -159.380 J mol -1 K -1, which indicated that OPA binds to BSA mainly by hydrogen bonds and van der Waals interactions. The binding distance between BSA and OPA was calculated to be 4.10 nm according to the theory of FÖrster's non-radiation energy transfer. The displacement experiments confirmed that OPA could bind to the site I of BSA. Furthermore, the effects of pH and some common ions on the binding constant were also examined. And the alterations of protein secondary structure in the presence of OPA were observed by the CD and FT-IR spectra.

Interaction of bovine serum albumin with N-acyl amino acid based anionic surfactants: Effect of head-group hydrophobicity.

PubMed

Ghosh, Subhajit; Dey, Joykrishna

2015-11-15

The function of a protein depends upon its structure and surfactant molecules are known to alter protein structure. For this reason protein-surfactant interaction is important in biological, pharmaceutical, and cosmetic industries. In the present work, interactions of a series of anionic surfactants having the same hydrocarbon chain length, but different amino acid head group, such as l-alanine, l-valine, l-leucine, and l-phenylalanine with the transport protein, bovine serum albumin (BSA), were studied at low surfactant concentrations using fluorescence and circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The results of fluorescence measurements suggest that the surfactant molecules bind simultaneously to the drug binding site I and II of the protein subdomain IIA and IIIA, respectively. The fluorescence as well as CD spectra suggest that the conformation of BSA goes to a more structured state upon surfactant binding at low concentrations. The binding constants of the surfactants were determined by the use of fluorescence as well as ITC measurements and were compared with that of the corresponding glycine-derived surfactant. The binding constant values clearly indicate a significant head-group effect on the BSA-surfactant interaction and the interaction is mainly hydrophobic in nature. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Sawada, Y.; Kawai, R.; McManaway, M.

(3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB);more » estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.« less

Diketo modification of curcumin affects its interaction with human serum albumin.

PubMed

Shaikh, Shaukat Ali M; Singh, Beena G; Barik, Atanu; Ramani, Modukuri V; Balaji, Neduri V; Subbaraju, Gottumukkala V; Naik, Devidas B; Indira Priyadarsini, K

2018-06-15

Curcumin isoxazole (CI) and Curcumin pyrazole (CP), the diketo modified derivatives of Curcumin (CU) are metabolically more stable and are being explored for pharmacological properties. One of the requirements in such activities is their interaction with circulatory proteins like human serum albumin (HSA). To understand this, the interactions of CI and CP with HSA have been investigated employing absorption and fluorescence spectroscopy and the results are compared with that of CU. The respective binding constants of CP, CI and CU with HSA were estimated to be 9.3×10 5 , 8.4×10 5 and 2.5×10 5 M -1 , which decreased with increasing salt concentration in the medium. The extent of decrease in the binding constant was the highest in CP followed by CI and CU. This revealed that along with hydrophobic interaction other binding modes like electrostatic interactions operate between CP/CI/CU with HSA. Fluorescence quenching studies of HSA with these compounds suggested that both static and dynamic quenching mechanisms operate, where the contribution of static quenching is higher for CP and CI than that for CU. From fluorescence resonance energy transfer studies, the binding site of CU, CI and CP was found to be in domain IIA of HSA. CU was found to bind in closer proximity with Trp214 as compared to CI and CP and the same was responsible for efficient energy transfer and the same was also established by fluorescence anisotropy measurements. Furthermore docking simulation complemented the experimental observation, where both electrostatic as well as hydrophobic interactions were indicated between HSA and CP, CI and CU. This study is useful in designing more stable CU derivatives having suitable binding properties with proteins like HSA. Copyright © 2018 Elsevier B.V. All rights reserved.

Diketo modification of curcumin affects its interaction with human serum albumin

NASA Astrophysics Data System (ADS)

Shaikh, Shaukat Ali M.; Singh, Beena G.; Barik, Atanu; Ramani, Modukuri V.; Balaji, Neduri V.; Subbaraju, Gottumukkala V.; Naik, Devidas B.; Indira Priyadarsini, K.

2018-06-01

Curcumin isoxazole (CI) and Curcumin pyrazole (CP), the diketo modified derivatives of Curcumin (CU) are metabolically more stable and are being explored for pharmacological properties. One of the requirements in such activities is their interaction with circulatory proteins like human serum albumin (HSA). To understand this, the interactions of CI and CP with HSA have been investigated employing absorption and fluorescence spectroscopy and the results are compared with that of CU. The respective binding constants of CP, CI and CU with HSA were estimated to be 9.3 × 105, 8.4 × 105 and 2.5 × 105 M-1, which decreased with increasing salt concentration in the medium. The extent of decrease in the binding constant was the highest in CP followed by CI and CU. This revealed that along with hydrophobic interaction other binding modes like electrostatic interactions operate between CP/CI/CU with HSA. Fluorescence quenching studies of HSA with these compounds suggested that both static and dynamic quenching mechanisms operate, where the contribution of static quenching is higher for CP and CI than that for CU. From fluorescence resonance energy transfer studies, the binding site of CU, CI and CP was found to be in domain IIA of HSA. CU was found to bind in closer proximity with Trp214 as compared to CI and CP and the same was responsible for efficient energy transfer and the same was also established by fluorescence anisotropy measurements. Furthermore docking simulation complemented the experimental observation, where both electrostatic as well as hydrophobic interactions were indicated between HSA and CP, CI and CU. This study is useful in designing more stable CU derivatives having suitable binding properties with proteins like HSA.

Ternary borate-nucleoside complex stabilization by Ribonuclease A demonstrates phosphate mimicry

PubMed Central

Gabel, Scott A.; London, Robert E.

2010-01-01

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate-ester recognition sites on proteins might exhibit significant affinity for non-enzymatically formed borate esters. 11B NMR studies and activity measurements on ribonuclease A in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A•3′-deoxycytidine-2′-borate ternary complex that mimics the complex formed between RNase A and a 2′-cytidine monophosphate (2′-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2′-deoxycytidine mixture, demonstrating the critical importance of the 2′-hydroxyl group for complex formation. Titration of the ternary complex with 2′-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2′CMP. RNase A binding of a cyclic cytidine-2′,3′-borate ester, which is a structural homolog of the cytidine-2′,3′-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2′,3′-borate•RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cCMP at pH 7, indicating considerably stronger binding. However, the value is 1000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters support enzyme catalysis. PMID:17957392

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters

PubMed Central

Hirayama, Bruce A; Díez-Sampedro, Ana; Wright, Ernest M

2001-01-01

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. PMID:11588102

A critical review of three methods used for the measurement of mercury (Hg2+)-dissolved organic matter stability constants

USGS Publications Warehouse

Gasper, J.D.; Aiken, G.R.; Ryan, J.N.

2007-01-01

Three experimental techniques - ion exchange, liquid-liquid extraction with competitive ligand exchange, and solid-phase extraction with competitive ligand exchange (CLE-SPE) - were evaluated as methods for determining conditional stability constants (K) for the binding of mercury (Hg2+) to dissolved organic matter (DOM). To determine the utility of a given method to measure stability constants at environmentally relevant experimental conditions, experimental results should meet three criteria: (1) the data must be experimentally valid, in that they were acquired under conditions that meet all the requirements of the experimental method, (2) the Hg:DOM ratio should be determined and it should fall within levels that are consistent with environmental conditions, and (3) the stability constants must fall within the detection window of the method. The ion exchange method was found to be limited by its detection window, which constrains the method to stability constants with log K values less than about 14. The liquid-liquid extraction method was found to be complicated by the ability of Hg-DOM complexes to partition into the organic phase. The CLE-SPE method was found to be the most suitable of these methods for the measurement of Hg-DOM stability constants. Stability constants for DOM isolates measured using the CLE-SPE method at environmentally relevant Hg:DOM ratios were log K = 25-30 (M-1). These values are consistent with the strong Hg2+ binding expected for reduced S-containing binding sites. ?? 2007 Elsevier Ltd. All rights reserved.

Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies

NASA Astrophysics Data System (ADS)

Śliwińska-Hill, Urszula

2017-02-01

Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 105 M- 1. The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn2 + and Ca2 + strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

PubMed

Henzl, Michael T; Markus, Lindsey A; Davis, Meredith E; McMillan, Andrew T

2013-03-01

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration. Copyright © 2013. Published by Elsevier Inc.

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach.

PubMed

Shamsi, Anas; Ahmed, Azaj; Khan, Mohd Shahnawaz; Husain, Fohad Mabood; Amani, Samreen; Bano, Bilqees

2018-05-16

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 10 4  M -1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes. Copyright © 2018 John Wiley & Sons, Ltd.

Structure and gelation properties of casein micelles doped with curcumin under acidic conditions.

PubMed

Khanji, Aya N; Michaux, Florentin; Jasniewski, Jordane; Petit, Jeremy; Lahimer, Emna; Cherif, Mohamed; Salameh, Dominique; Rizk, Toufic; Banon, Sylvie

2015-12-01

In this study, the ability of micellar casein (MC) to interact with curcumin during acidification and to produce acid gel was investigated. Steady-state fluorescence spectroscopy of curcumin variation and fluorescence quenching of caseins upon binding with curcumin molecules were evidenced. Increasing the temperature from 20 to 35 °C enhanced MC-curcumin interactions as reflected by the increase in the binding constant from 0.6 ± 0.3 × 10(4) to 6.6 ± 0.6 × 10(4) M(-1). From changes in entropy, enthalpy and Gibbs free energy, hydrophobic interactions were proposed as major binding forces. Static fluorescence MC quenching was demonstrated for the MC-curcumin complex during acidification. From pH 7.4 to pH 5.0, the binding site numbers varied in the range from 1.25 ± 0.05 to 1.49 ± 0.05 and the binding constant kb varied from 3.9 ± 0.4 × 10(4) to 7.5 ± 0.7 × 10(4) M(-1). Small angle X-ray scattering profiles demonstrated that the MC internal structure was unchanged upon curcumin binding. The ζ-potential value of curcumin-doped MC indicated that curcumin did not modify the global charge of MC particles. Acid gelation studied by oscillation rheology and static multiple light scattering at 20 and 35 °C led to a similar behavior for native and curcumin-doped MC suspensions. For the first time, it was demonstrated that the colloidal and functional properties of MC were unchanged when doped with curcumin during acidification.

Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy.

PubMed

Hur, Jin; Lee, Bo-Mi

2011-06-01

The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.

PubMed

Hou, Yuting; An, Jianhong; Deng, Chunyan; Chen, Shu; Xiang, Juan

2016-07-01

The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt c red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.

Insights into in vitro binding of parecoxib to human serum albumin by spectroscopic methods.

PubMed

Shang, Shujun; Liu, Qingling; Gao, Jiandong; Zhu, Yulin; Liu, Jingying; Wang, Kaiyan; Shao, Wei; Zhang, Shudong

2014-10-01

Herein, we report the effect of parecoxib on the structure and function of human serum albumin (HSA) by using fluorescence, circular dichroism (CD), Fourier transforms infrared (FTIR), three-dimensional (3D) fluorescence spectroscopy, and molecular docking techniques. The Stern-Volmer quenching constants K(SV) and the corresponding thermodynamic parameters ΔH, ΔG, and ΔS have been estimated by the fluorescence quenching method. The results indicated that parecoxib binds spontaneously with HSA through van der Waals forces and hydrogen bonds with binding constant of 3.45 × 10(4) M(-1) at 298 K. It can be seen from far-UV CD spectra that the α-helical network of HSA is disrupted and its content decreases from 60.5% to 49.6% at drug:protein = 10:1. Protein tertiary structural alterations induced by parecoxib were also confirmed by FTIR and 3D fluorescence spectroscopy. The molecular docking study indicated that parecoxib is embedded into the hydrophobic pocket of HSA. © 2014 Wiley Periodicals, Inc.

ELECTRON STAINS

PubMed Central

Zobel, C. Richard; Beer, Michael

1961-01-01

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO2++/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy. PMID:13788706

Spectroscopic study of the interaction of styrylcyanine dyes Sbo, Sil and their derivatives with bovine serum albumin.

PubMed

Kurtaliev, Eldar N

2011-07-01

The spectral-luminescent characteristics of newly synthesized styrylcyanine dyes on the base of dyes Sbo ((E)-2-(4-(dimethylamino)styryl)-3-methylbenzo[d]oxazol-3-ium iodide) and Sil ((E)-2-(4-(dimethylamino)styryl)-1,3,3-trimethyl-3H-indolium perchlorate) in aqueous solutions without and in the presence of bovine serum albumin (BSA) were studied. It was established that the absorption spectra of dyes Tol-6, Dbo-10 and Dil-10 with increasing amount of BSA appear new bands with λ(max)=505 nm, λ(max)=512 nm and λ(max)=566 nm, respectively, whose intensity increases in proportion to the amount of albumin. The intensity of the glow of the main band of fluorescence in the presence of BSA sharply increases. The binding constant (K) and the number of binding sites (N) of studied dyes with BSA were determined. The dependence of binding constants with BSA on the dipole moment of dye molecules was determined, which indicates that besides electrostatic forces of attraction between molecules styrylcyanine dyes with BSA, hydrophobic interactions are essential. © Springer Science+Business Media, LLC 2011

Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

NASA Astrophysics Data System (ADS)

Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

2008-11-01

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

PubMed

Acharya, Bipul R; Choudhury, Diptiman; Das, Amlan; Chakrabarti, Gopal

2009-07-28

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Adsorption of surfactant ions and binding of their counterions at an air/water interface.

PubMed

Tagashira, Hiroaki; Takata, Youichi; Hyono, Atsushi; Ohshima, Hiroyuki

2009-01-01

An expression for the surface tension of an aqueous mixed solution of surfactants and electrolyte ions in the presence of the common ions was derived from the Helmholtz free energy of an air/water surface. By applying the equation to experimental data for the surface tension, the adsorption constant of surfactant ions onto the air/water interface, the binding constant of counterions on the surfactants, and the surface potential and surface charge density of the interface were estimated. The adsorption constant and binding constant were dependent on the species of surfactant ion and counterion, respectively. Taking account of the dependence of surface potential and surface charge density on the concentration of electrolyte, it was suggested that the addition of electrolyte to the aqueous surfactant solution brings about the decrease in the surface potential, the increase in the surface density of surfactant ions, and consequently, the decrease in the surface tension. Furthermore, it was found that the configurational entropy plays a predominant role for the surface tension, compared to the electrical work.

Integrating a Smartphone and Molecular Modeling for Determining the Binding Constant and Stoichiometry Ratio of the Iron(II)-Phenanthroline Complex: An Activity for Analytical and Physical Chemistry Laboratories

ERIC Educational Resources Information Center

de Morais, Camilo de L. M.; Silva, Se´rgio R. B.; Vieira, Davi S.; Lima, Ka´ssio M. G.

2016-01-01

The binding constant and stoichiometry ratio for the formation of iron(II)-(1,10-phenanthroline) or iron(II)-o-phenanthroline complexes has been determined by a combination of a low-cost analytical method using a smartphone and a molecular modeling method as a laboratory experiment designed for analytical and physical chemistry courses. Intensity…

Competitive counterion complexation allows the true host : guest binding constants from a single titration by ionic receptors.

PubMed

Pessêgo, Márcia; Basílio, Nuno; Muñiz, M Carmen; García-Río, Luis

2016-07-06

Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration.

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC(50) values for the 6 frequently used anesthetics in GLIC for the site identified in the experimental crystal data (P = 0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these 6 anesthetics' known experimental EC(50) values. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (AutoDock) for both water-soluble and membrane proteins. Correlation of predicted affinity and EC(50) for 6 frequently used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism.

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring.

PubMed

Dumortier, C; Potenziano, J L; Bane, S; Engelborghs, Y

1997-10-01

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC50s for the six commonly used anesthetics in GLIC for the site identified in the experimental crystal data (p=0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these six anesthetics’ known experimental EC50s. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. Conclusion We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (Autodock) for both water soluble and membrane proteins. Correlation of predicted affinity and EC50 for six commonly used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism. PMID:22392968

Fulvic acid-sulfide ion competition for mercury ion binding in the Florida everglades

USGS Publications Warehouse

Reddy, M.M.; Aiken, G.R.

2001-01-01

Negatively charged functional groups of fulvic acid compete with inorganic sulfide ion for mercury ion binding. This competition is evaluated here by using a discrete site-electrostatic model to calculate mercury solution speciation in the presence of fulvic acid. Model calculated species distributions are used to estimate a mercury-fulvic acid apparent binding constant to quantify fulvic acid and sulfide ion competition for dissolved inorganic mercury (Hg(II)) ion binding. Speciation calculations done with PHREEQC, modified to use the estimated mercury-fulvic acid apparent binding constant, suggest that mercury-fulvic acid and mercury-sulfide complex concentrations are equivalent for very low sulfide ion concentrations (about 10-11 M) in Everglades' surface water. Where measurable total sulfide concentration (about 10-7 M or greater) is present in Everglades' surface water, mercury-sulfide complexes should dominate dissolved inorganic mercury solution speciation. In the absence of sulfide ion (for example, in oxygenated Everglades' surface water), fulvic acid binding should dominate Everglades' dissolved inorganic mercury speciation.

Protocols Utilizing Constant pH Molecular Dynamics to Compute pH-Dependent Binding Free Energies

PubMed Central

2015-01-01

In protein–ligand binding, the electrostatic environments of the two binding partners may vary significantly in bound and unbound states, which may lead to protonation changes upon binding. In cases where ligand binding results in a net uptake or release of protons, the free energy of binding is pH-dependent. Nevertheless, conventional free energy calculations and molecular docking protocols typically do not rigorously account for changes in protonation that may occur upon ligand binding. To address these shortcomings, we present a simple methodology based on Wyman’s binding polynomial formalism to account for the pH dependence of binding free energies and demonstrate its use on cucurbit[7]uril (CB[7]) host–guest systems. Using constant pH molecular dynamics and a reference binding free energy that is taken either from experiment or from thermodynamic integration computations, the pH-dependent binding free energy is determined. This computational protocol accurately captures the large pKa shifts observed experimentally upon CB[7]:guest association and reproduces experimental binding free energies at different levels of pH. We show that incorrect assignment of fixed protonation states in free energy computations can give errors of >2 kcal/mol in these host–guest systems. Use of the methods presented here avoids such errors, thus suggesting their utility in computing proton-linked binding free energies for protein–ligand complexes. PMID:25134690

Drug-DNA interactions at single molecule level: A view with optical tweezers

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan

Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by stretching the DNA molecule to a certain force and holding it at constant force while adding the drug and then while washing off the drug. Our finding resolves the long lasting controversy of ActD binding modes, clearly showing that both the dsDNA binding and ssDNA binding converge to the same single mode. The result supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by binding to transcription bubbles and causing cell death.

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

Binding of Phenazinium Dye Safranin T to Polyriboadenylic Acid: Spectroscopic and Thermodynamic Study

PubMed Central

Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na+] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure. PMID:24498422

Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

PubMed

Pradhan, Ankur Bikash; Haque, Lucy; Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

α‐Conotoxin M1 (CTx) blocks αδ binding sites of adult nicotinic receptors while ACh binding at αε sites elicits only small and short quantal synaptic currents

PubMed Central

Dudel, Josef

2014-01-01

Abstract In ‘embryonic’ nicotinic receptors, low CTx concentrations are known to block only the αδ binding site, whereas binding of ACh at the αγ‐site elicits short single channel openings and short bursts. In adult muscles the αγ‐ is replaced by the αε‐site. Quantal EPSCs (qEPSCs) were elicited in adult muscles by depolarization pulses and recorded through a perfused macropatch electrode. One to 200 nmol L−1 CTx reduced amplitudes and decay time constants of qEPSCs, but increased their rise times. CTx block at the αδ binding sites was incomplete: The qEPSCs still contained long bursts from not yet blocked receptors, whereas their average decay time constants were reduced by a short burst component generated by ACh binding to the αε‐site. Two nanomolar CTx applied for 3 h reduced the amplitudes of qEPSCs to less than half with a constant slope. The equilibrium concentration of the block is below 1 nmol L−1 and lower than that of embryonic receptors. CTx‐block increased in proportion to CTx concentrations (average rate 2 × 104 s−1·mol−1 L). Thus, the reactions of ‘embryonic’ and of adult nicotinic receptors to block by CTx are qualitatively the same. – The study of the effects of higher CTx concentrations or of longer periods of application of CTx was limited by presynaptic effects of CTx. Even low CTx concentrations severely reduced the release of quanta by activating presynaptic M2 receptors at a maximal rate of 6 × 105 s−1·mol−1 L. When this dominant inhibition was prevented by blocking the M2 receptors with methoctramine, activation of M1 receptors was unmasked and facilitated release. PMID:25501436

Anticancer drug-DNA interactions measured using a photoinduced electron-transfer mechanism based on luminescent quantum dots.

PubMed

Yuan, Jipei; Guo, Weiwei; Yang, Xiurong; Wang, Erkang

2009-01-01

A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L(-1) and a linear detection range from 10 nmol L(-1) to 4.5 micromol L(-1) was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

Binding interaction between rice glutelin and amylose: Hydrophobic interaction and conformational changes.

PubMed

Xu, Xingfeng; Liu, Wei; Zhong, Junzhen; Luo, Liping; Liu, Chengmei; Luo, Shunjing; Chen, Lin

2015-11-01

The interaction of rice glutelin (RG) with amylose was characterized by spectroscopic and molecular docking studies. The intrinsic fluorescence of RG increased upon the addition of amylose. The binding sites, binding constant and thermodynamic features indicated that binding process was spontaneous and the main driving force of the interaction was hydrophobic interaction. The surface hydrophobicity of RG decreased with increasing amount of amylose. Furthermore, synchronous fluorescence and circular dichroism (CD) spectra provided data concerning conformational and micro-environmental changes of RG. With the concentration of amylose increasing, the polarity around the tyrosine residues increased while the hydrophobicity decreased. Alteration of protein conformation was observed with increasing of α-helix and reducing of β-sheet. Finally, a visual representation of two binding sites located in the amorphous area of RG was presented by molecular modeling studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Hydrogen bonding-assisted interaction between amitriptyline hydrochloride and hemoglobin: spectroscopic and molecular dynamics studies.

PubMed

Maurya, Neha; Maurya, Jitendra Kumar; Kumari, Meena; Khan, Abbul Bashar; Dohare, Ravins; Patel, Rajan

2017-05-01

Herein, we have explored the interaction between amitriptyline hydrochloride (AMT) and hemoglobin (Hb), using steady-state and time-resolved fluorescence spectroscopy, UV-visible spectroscopy, and circular dichroism spectroscopy, in combination with molecular docking and molecular dynamic (MD) simulation methods. The steady-state fluorescence reveals the static quenching mechanism in the interaction system, which was further confirmed by UV-visible and time-resolved fluorescence spectroscopy. The binding constant, number of binding sites, and thermodynamic parameters viz. ΔG, ΔH, ΔS are also considered; result confirms that the binding of the AMT with Hb is a spontaneous process, involving hydrogen bonding and van der Waals interactions with a single binding site, as also confirmed by molecular docking study. Synchronous fluorescence, CD data, and MD simulation results contribute toward understanding the effect of AMT on Hb to interpret the conformational change in Hb upon binding in aqueous solution.

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus.

PubMed Central

Meisch, H U; Schmitt, J A

1986-01-01

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of KD = 1.59 X 10 M (pKD = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals. From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed. PMID:3709455

Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

NASA Astrophysics Data System (ADS)

Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

2010-01-01

The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

DNA-Binding Interaction Studies of Microwave Assisted Synthesized Sulfonamide Substituted 8-Hydroxyquinoline Derivatives.

PubMed

Dixit, Ritu B; Patel, Tarosh S; Vanparia, Satish F; Kunjadiya, Anju P; Keharia, Harish R; Dixit, Bharat C

2011-01-01

Sulfonamide substituted 8-hydroxyquinoline derivatives were prepared using a microwave synthesizer. The interaction of sulfonamide substituted 8-hydroxyquinoline derivatives and their transition metal complexes with Plasmid (pUC 19) DNA and Calf Thymus DNA were investigated by UV spectroscopic studies and gel electrophoresis measurements. The interaction between ligand/metal complexes and DNA was carried out by increasing the concentration of DNA from 0 to 12 μl in UV spectroscopic study, while the concentration of DNA in gel electrophoresis remained constant at 10 μl. These studies supported the fact that, the complex binds to DNA by intercalation via ligand into the base pairs of DNA. The relative binding efficacy of the complexes to DNA was much higher than the binding efficacy of ligands, especially the complex of Cu-AHQMBSH had the highest binding ability to DNA. The mobility of the bands decreased as the concentration of the complex was increased, indicating that there was increase in the interaction between the metal ion and DNA. Complexes of AHQMBSH were excellent for DNA binding as compared to HQMABS.

Critical insight into the interaction of naringenin with human haemoglobin: A combined spectroscopic and computational modeling approaches

NASA Astrophysics Data System (ADS)

Maity, Subhajit; Chakraborty, Sandipan; Chakraborti, Abhay Sankar

2017-02-01

The present study demonstrates critical insight into the binding of a bioactive flavanone naringenin with normal human haemoglobin (NHb). Both spectrophotometric and spectrofluorimetric studies reveal that naringenin interacts with NHb. The binding affinity constant and number of binding sites appear to be approximately (1.5 ± 0.2) × 104 M-1 and 1, respectively. Static quenching seems to be an important factor in binding process, as evident from steady-state and time-resolved fluorescence spectroscopic studies. Far UV circular dichroism spectroscopy depicts that binding of naringenin to NHb causes no change in the secondary structure of the protein, which is also evident from Fourier transform infrared spectroscopic study. Free energy change (ΔG0) for naringenin-NHb interaction, determined by spectroscopic and isothermal calorimetric method, appears to be -5.67 kcal/mol and -6.90 kcal/mol, respectively, and is close to the docking energy -6.84 kcal/mol. Molecular docking suggests that naringenin binds near the cavity of the tetrameric heme protein, forming hydrogen bonds with surrounding amino acid residues. The binding site is away from the heme moieties, implicating naringenin binding does not affect the oxygen binding capacity of NHb, which makes the protein a suitable carrier of the flavonoid.

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

PubMed

Volkán-Kacsó, Sándor; Marcus, Rudolph A

2016-10-25

A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

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

D'Aquino,J.; Tetenbaum-Novatt, J.; White, A.

2005-01-01

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10{sup -7}, binding site 2 (primary) is a low-affinity binding site with amore » binding constant of 6.3 x 10{sup -4}. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A, C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.« less

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin

PubMed Central

FEROZ, Shevin R.; SUMI, Rumana A.; MALEK, Sri N.A.; TAYYAB, Saad

2014-01-01

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, Ka in the range of 1.49 – 6.12 × 104 M−1, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics. PMID:25519455

Biochemical activity of a fluorescent dye rhodamine 6G: Molecular modeling, electrochemical, spectroscopic and thermodynamic studies.

PubMed

Al Masum, Abdulla; Chakraborty, Maharudra; Ghosh, Soumen; Laha, Dipranjan; Karmakar, Parimal; Islam, Md Maidul; Mukhopadhyay, Subrata

2016-11-01

Interaction of CT DNA with Rhodamine 6G (R6G) has been studied using molecular docking, electrochemical, spectroscopic and thermodynamic methods. From the study, it was illustrated that Rhodamine 6G binds to the minor groove of CT DNA. The binding was cooperative in nature. Circular voltametric study showed significant change in peak current and peak potential due to complexation. All the studies showed that the binding constant was in the order of 10 6 M -1 . Circular dichroic spectra showed significant conformational change on binding and DNA unwind during binding. Thermodynamic study showed that binding was favored by negative enthalpy and positive entropy change. From thermodynamic study it was also observed that several positive and negative free energies played significant role during binding and the unfavorable conformational free energy change was overcame by highly negative hydrophobic and salt dependent free energy changes. The experimental results were further validated using molecular docking study and the effect of structure on binding has been studied theoretically. From docking study it was found that the hydrophobic interaction and hydrogen bonds played a significant role during binding. The dye was absorbed by cell and this phenomenon was studied using fluorescent microscope. Cell survivability test showed that the dye active against Human Breast Cancer cells MDA-MB 468. ROS study showed that the activity is due to the production of reactive oxygen. Copyright © 2016 Elsevier B.V. All rights reserved.

Spectroscopic and electrochemical studies of the interaction between oleuropein, the major bio-phenol in olives, and salmon sperm DNA

NASA Astrophysics Data System (ADS)

Mohamadi, Maryam; Afzali, Daryoush; Esmaeili-Mahani, Saeed; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2015-09-01

Interaction of oleuropein, the major bio-phenol in olive leaf and fruit, with salmon sperm double-stranded DNA was investigated by employing electronic absorption titrations, fluorescence quenching spectroscopy, competitive fluorescence spectroscopy, thermal denaturation and voltammetric studies. Titration of oleuropein with the DNA caused a hypochromism accompanied with a red shift indicating an intercalative mode of interaction. Binding constant of 1.4 × 104 M-1 was obtained for this interaction. From the curves of fluorescence titration of oleuropein with the DNA, binding constant and binding sites were calculated to be 8.61 × 103 M-1 and 1.05, respectively. Competitive studies with ethidium bromide (a well-known DNA intercalator) showed that the bio-phenol could take the place of ethidium bromide in the DNA intercalation sites. The interaction of oleuropein with DNA was also studied electrochemically. In the presence of the DNA, the anodic and cathodic peak currents of oleuropein decreased accompanied with increases in peak-to-peak potential separation and formal potential, indicating the intercalation of oleuropein into the DNA double helix. Moreover, melting temperature of the DNA was found to increase in the presence of oleuropein, indicating the stabilization of the DNA double helix due to an intercalative interaction.

Characterization of the Interaction between Gallic Acid and Lysozyme by Molecular Dynamics Simulation and Optical Spectroscopy

PubMed Central

Zhan, Minzhong; Guo, Ming; Jiang, Yanke; Wang, Xiaomeng

2015-01-01

The binding interaction between gallic acid (GA) and lysozyme (LYS) was investigated and compared by molecular dynamics (MD) simulation and spectral techniques. The results from spectroscopy indicate that GA binds to LYS to generate a static complex. The binding constants and thermodynamic parameters were calculated. MD simulation revealed that the main driving forces for GA binding to LYS are hydrogen bonding and hydrophobic interactions. The root-mean-square deviation verified that GA and LYS bind to form a stable complex, while the root-mean-square fluctuation results showed that the stability of the GA-LYS complex at 298 K was higher than that at 310 K. The calculated free binding energies from the molecular mechanics/Poisson-Boltzmann surface area method showed that van der Waals forces and electrostatic interactions are the predominant intermolecular forces. The MD simulation was consistent with the spectral experiments. This study provides a reference for future study of the pharmacological mechanism of GA. PMID:26140374

Characterization of the Interaction between Gallic Acid and Lysozyme by Molecular Dynamics Simulation and Optical Spectroscopy.

PubMed

Zhan, Minzhong; Guo, Ming; Jiang, Yanke; Wang, Xiaomeng

2015-07-01

The binding interaction between gallic acid (GA) and lysozyme (LYS) was investigated and compared by molecular dynamics (MD) simulation and spectral techniques. The results from spectroscopy indicate that GA binds to LYS to generate a static complex. The binding constants and thermodynamic parameters were calculated. MD simulation revealed that the main driving forces for GA binding to LYS are hydrogen bonding and hydrophobic interactions. The root-mean-square deviation verified that GA and LYS bind to form a stable complex, while the root-mean-square fluctuation results showed that the stability of the GA-LYS complex at 298 K was higher than that at 310 K. The calculated free binding energies from the molecular mechanics/Poisson-Boltzmann surface area method showed that van der Waals forces and electrostatic interactions are the predominant intermolecular forces. The MD simulation was consistent with the spectral experiments. This study provides a reference for future study of the pharmacological mechanism of GA.

Mercury(II) sorption to two Florida Everglades peat--Evidence for strong and weak binding and competition by dissolved organic matter released from the peat

USGS Publications Warehouse

Drexel, R. Todd; Haitzer, Markus; Ryan, Joseph N.; Aiken, George R.; Nagy, Kathryn L.

2002-01-01

The binding of mercury(II) to two peats from Florida Everglades sites with different rates of mercury methylation was measured at pH 6.0 and 0.01 M ionic strength. The mercury(II) sorption isotherms, measured over a total mercury(II) range of 10-7.4 to 10-3.7 M, showed the competition for mercury(II) between the peat and dissolved organic matter released from the peat and the existence of strong and weak binding sites for mercury(II). Binding was portrayed by a model accounting for strong and weak sites on both the peat and the released DOM. The conditional binding constants (for which the ligand concentration was set as the concentration of reduced sulfur in the organic matter as measured by X-ray absorption near-edge structure spectroscopy) determined for the strong sites on the two peats were similar (Kpeat,s = 1021.8±0.1and 1022.0±0.1 M-1), but less than those determined for the DOM strong sites (Kdom,s = 1022.8±0.1and 1023.2±0.1 M-1), resulting in mercury(II) binding by the DOM at low mercury(II) concentrations. The magnitude of the strong site binding constant is indicative of mercury(II) interaction with organic thiol functional groups. The conditional binding constants determined for the weak peat sites (Kpeat,w = 1011.5±0.1 and 1011.8±0.1 M-1) and weak DOM sites (Kdom,w = 108.7±3.0 and 107.3±4.5 M-1) were indicative of mercury(II) interaction with carboxyl and phenol functional groups.

Prediction of binding constants of protein ligands: A fast method for the prioritization of hits obtained from de novo design or 3D database search programs

NASA Astrophysics Data System (ADS)

Böhm, Hans-Joachim

1998-07-01

A dataset of 82 protein-ligand complexes of known 3D structure and binding constant Ki was analysed to elucidate the important factors that determine the strength of protein-ligand interactions. The following parameters were investigated: the number and geometry of hydrogen bonds and ionic interactions between the protein and the ligand, the size of the lipophilic contact surface, the flexibility of the ligand, the electrostatic potential in the binding site, water molecules in the binding site, cavities along the protein-ligand interface and specific interactions between aromatic rings. Based on these parameters, a new empirical scoring function is presented that estimates the free energy of binding for a protein-ligand complex of known 3D structure. The function distinguishes between buried and solvent accessible hydrogen bonds. It tolerates deviations in the hydrogen bond geometry of up to 0.25 Å in the length and up to 30 °Cs in the hydrogen bond angle without penalizing the score. The new energy function reproduces the binding constants (ranging from 3.7 × 10-2 M to 1 × 10-14 M, corresponding to binding energies between -8 and -80 kJ/mol) of the dataset with a standard deviation of 7.3 kJ/mol corresponding to 1.3 orders of magnitude in binding affinity. The function can be evaluated very fast and is therefore also suitable for the application in a 3D database search or de novo ligand design program such as LUDI. The physical significance of the individual contributions is discussed.

Temperature-Dependent Estimation of Gibbs Energies Using an Updated Group-Contribution Method.

PubMed

Du, Bin; Zhang, Zhen; Grubner, Sharon; Yurkovich, James T; Palsson, Bernhard O; Zielinski, Daniel C

2018-06-05

Reaction-equilibrium constants determine the metabolite concentrations necessary to drive flux through metabolic pathways. Group-contribution methods offer a way to estimate reaction-equilibrium constants at wide coverage across the metabolic network. Here, we present an updated group-contribution method with 1) additional curated thermodynamic data used in fitting and 2) capabilities to calculate equilibrium constants as a function of temperature. We first collected and curated aqueous thermodynamic data, including reaction-equilibrium constants, enthalpies of reaction, Gibbs free energies of formation, enthalpies of formation, entropy changes of formation of compounds, and proton- and metal-ion-binding constants. Next, we formulated the calculation of equilibrium constants as a function of temperature and calculated the standard entropy change of formation (Δ f S ∘ ) using a model based on molecular properties. The median absolute error in estimating Δ f S ∘ was 0.013 kJ/K/mol. We also estimated magnesium binding constants for 618 compounds using a linear regression model validated against measured data. We demonstrate the improved performance of the current method (8.17 kJ/mol in median absolute residual) over the current state-of-the-art method (11.47 kJ/mol) in estimating the 185 new reactions added in this work. The efforts here fill in gaps for thermodynamic calculations under various conditions, specifically different temperatures and metal-ion concentrations. These, to our knowledge, new capabilities empower the study of thermodynamic driving forces underlying the metabolic function of organisms living under diverse conditions. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

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

Giannopoulos, G.; Jackson, K.; Kredentser, J.

The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

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

Ayyagari, R.R.; Khan-Dawood, F.S.

Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2more » hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.« less

Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): With the aim of the drug interactions probing

NASA Astrophysics Data System (ADS)

Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

2015-02-01

The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy.

Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): with the aim of the drug interactions probing.

PubMed

Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

2015-02-25

The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy. Copyright © 2014 Elsevier B.V. All rights reserved.

Binding study of lysozyme with Al(III) using chemiluminescence analysis.

PubMed

Liu, Jiangman; Luo, Kai; Song, Zhenghua

2014-09-01

The binding behavior of lysozyme with Al(III) is described using luminol as a luminescence probe by flow injection-chemiluminescence (FI-CL) analysis. It was found that the CL intensity of the luminol-lysozyme reaction could be markedly enhanced by Al(III), and the increase in CL intensity was linear with the Al(III) concentration over the range 0.3-30.0  pg  mL(-1) , with a detection limit of 0.1 pg  mL(-1) (3σ). Based on the interaction model of lysozyme with Al(III), lg[(I - I0 )/(2I0  - I)] = lgK + nlg[M], the binding constant K = 6.84 × 10(6)  L mol(-1) and the number of binding sites (n) = 0.76. The relative standard deviations were 3.2, 2.4 and 2.0% for 10.0, 20.0 and 30.0  pg  mL(-1) Al(III) (n = 7), respectively. This new method was successfully applied to continuous, quantitative monitoring of picogram level Al(III) in human saliva following oral intake of compound aluminum hydroxide tablets. It was found that Al(III) in saliva reached a maximum of 101.2  ng  mL(-1) at 3.0 h. The absorption rate constant ka , elimination rate constant k and half-life time t1/2 of Al(III) were 1.378  h(-1) , 0.264  h(-1) and 2.624  h, respectively. Copyright © 2013 John Wiley & Sons, Ltd.

EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc.

PubMed

Waters, R S; Bryden, N A; Patterson, K Y; Veillon, C; Anderson, R A

2001-12-01

The efficacy of a chelating agent in binding a given metal in a biological system depends on the binding constants of the chelator for the particular metals in the system, the concentration of the metals, and the presence and concentrations of other ligands competing for the metals in question. In this study, we make a comparison of the in vitro binding constants for the chelator, ethylenediaminetetraacetic acid, with the quantitative urinary excretion of the metals measured before and after EDTA infusion in 16 patients. There were significant increases in lead, zinc, cadmium, and calcium, and these increases roughly corresponded to the expected relative increases predicted by the EDTA-metal-binding constants as measured in vitro. There were no significant increases in urinary cobalt, chromium, or copper as a result of EDTA infusion. The actual increase in cobalt could be entirely attributed to the cobalt content of the cyanocobalamin that was added to the infusion. Although copper did increase in the post-EDTA specimens, the increase was not statistically significant. In the case of magnesium, there was a net retention of approximately 85% following chelation. These data demonstrate that EDTA chelation therapy results in significantly increased urinary losses of lead, zinc, cadmium, and calcium following EDTA chelation therapy. There were no significant changes in cobalt, chromium, or copper and a retention of magnesium. These effects are likely to have significant effects on nutrient concentrations and interactions and partially explain the clinical improvements seen in patients undergoing EDTA chelation therapy.

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

PubMed Central

Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

2014-01-01

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases. PMID:25420047

Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

PubMed

Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

2014-01-01

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

PubMed Central

Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

2012-01-01

Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrP(C) to PrP(Sc).

PubMed

Pagadala, Nataraj S; Perez-Pineiro, Rolando; Wishart, David S; Tuszynski, Jack A

2015-02-16

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 μM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 μM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between β2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while noscapine might act as a prion accelerator from PrP(C) to PrP(Sc). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

The effect of Cu(2+) chelation on the direct photolysis of oxytetracycline: A study assisted by spectroscopy analysis and DFT calculation.

PubMed

Jin, Xin; Qiu, Shanshan; Wu, Ke; Jia, Mingyun; Wang, Fang; Gu, Chenggang; Zhang, Aiqian; Jiang, Xin

2016-07-01

The extensive usage of OTC and Cu(2+) in livestock and poultry industry caused high residues in natural environment. Co-contamination of OTC and Cu(2+) was a considerable environmental problem in surface waters. In this study, Cu(2+) mediated direct photolysis of OTC was studied. Cu(2+) chelating with OTC was found to greatly inhibit OTC photodegradation. To reveal the chelation mechanism of OTC-Cu complexes, multiple methods including UV-Vis absorption spectra, Infrared (IR) spectra, mass spectroscopy, and density functional theoretical (DFT) modeling were performed. Four OTC-Cu complexes were proposed. Cu(2+) preferably bond to O11O12 site with the binding constants logK = 8.19 and 7.86 for CuHL+ and CuL±, respectively. The second chelating site was suggested to be O2O3 with the binding constants of logK = 4.41 and 4.62 for Cu2HL3+ and Cu2L2+, respectively. The suppressed quantum yield of OTC by Cu2+ chelation was accused for their intra-/inter-molecular electron transfer, by which the energy in activated states was distributed. The occurrence of electron transfer between BCD ring and A ring also from BCD ring to Cu was evidenced by the TD-DFT result only for the OTC-Cu complexes. Besides, the cyclic voltammetry measurement also suggested one OTC-Cu(II)/OTC-Cu(I) redox couple. These results suggested that the persistence of OTC in environmental surface waters will probably be underestimated for neglecting the chelating effect of Cu2+. The photolysis quantum yield of OTC-Cu complexes, as well as the specific molar absorption constants, the equilibrium binding constants of Cu2+ with OTC could contribute to more accurate kinetic models of OTC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

ERIC Educational Resources Information Center

Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

2010-01-01

The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

NASA Astrophysics Data System (ADS)

Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

2017-09-01

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

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.

Comparison of calculation and experiment implicates significant electrostatic contributions to the binding stability of barnase and barstar.

PubMed

Dong, Feng; Vijayakumar, M; Zhou, Huan-Xiang

2003-07-01

The contributions of electrostatic interactions to the binding stability of barnase and barstar were studied by the Poisson-Boltzmann model with three different protocols: a), the dielectric boundary specified as the van der Waals (vdW) surface of the protein along with a protein dielectric constant (epsilon (p)) of 4; b), the dielectric boundary specified as the molecular (i.e., solvent-exclusion (SE)) surface along with epsilon (p) = 4; and c), "SE + epsilon (p) = 20." The "vdW + epsilon (p) = 4" and "SE + epsilon (p) = 20" protocols predicted an overall electrostatic stabilization whereas the "SE + epsilon (p) = 4" protocol predicted an overall electrostatic destabilization. The "vdW + epsilon (p) = 4" protocol was most consistent with experiment. It quantitatively reproduced the observed effects of 17 mutations neutralizing charged residues lining the binding interface and the measured coupling energies of six charge pairs across the interface and reasonably rationalized the experimental ionic strength and pH dependences of the binding constant. In contrast, the "SE + epsilon (p) = 4" protocol predicted significantly larger coupling energies of charge pairs whereas the "SE + epsilon (p) = 20" protocol did not predict any pH dependence. This study calls for further scrutiny of the different Poisson-Boltzmann protocols and demonstrates potential danger in drawing conclusions on electrostatic contributions based on a particular calculation protocol.

A comparative study on the binding of single and double chain surfactant-cobalt(III) complexes with bovine serum albumin

NASA Astrophysics Data System (ADS)

Vignesh, G.; Sugumar, K.; Arunachalam, S.; Vignesh, S.; Arthur James, R.

2013-09-01

The comparative binding effect of single and double aliphatic chain containing surfactant-cobalt(III) complexes cis-[Co(bpy)2(DA)2](ClO4)3ṡ2H2O (1), cis-[Co(bpy)2(DA)Cl](ClO4)2ṡ2H2O (2), cis-[Co(phen)2(CA)2](ClO4)3ṡ2H2O (3), and cis-[Co(phen)2(CA)Cl](ClO4)2ṡ2H2O (4) with bovine serum albumin (BSA) under physiological condition was analyzed by steady state, time resolved fluorescence, synchronous, three-dimensional fluorescence, UV-Visible absorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of BSA through a static mechanism. The binding constants (Kb) and the number of binding sites were calculated and binding constant values are found in the range of 104-105 M-1. The results indicate that compared to single chain complex, double chain surfactant-cobalt(III) complex interacts strongly with BSA. Also the sign of thermodynamic parameters (ΔG°, ΔH°, and ΔS°) indicate that all the complexes interact with BSA through hydrophobic force. The binding distance (r) between complexes and BSA was calculated using Förster non-radiation energy transfer theory and found to be less than 7 nm. The results of synchronous, three dimensional fluorescence and circular dichroism spectroscopic methods indicate that the double chain surfactant-cobalt(III) complexes changed the conformation of the protein considerably than the respective single chain surfactant-cobalt(III) complexes. Antimicrobial studies of the complexes showed good activities against pathogenic microorganisms.

Method bacterial endospore quantification using lanthanide dipicolinate luminescence

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J. (Inventor); Kirby, James Patrick (Inventor); Ponce, Adrian (Inventor)

2007-01-01

A lanthanide is combined with a medium to be tested for endospores. The dipicolinic acid released from the endospores binds the lanthanides, which have distinctive emission (i.e., luminescence) spectra, and are detected using photoluminescence. The concentration of spores is determined by preparing a calibration curve generated from photoluminescence spectra of lanthanide complex mixed with spores of a known concentration. A lanthanide complex is used as the analysis reagent, and is comprised of lanthanide ions bound to multidentate ligands that increase the dipicolinic acid binding constant through a cooperative binding effect with respect to lanthanide chloride. The resulting combined effect of increasing the binding constant and eliminating coordinated water and multiple equilibria increase the sensitivity of the endospore assay by an estimated three to four orders of magnitude over prior art of endospore detection based on lanthanide luminescence.

Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

PubMed

Giri, Jyotsnendu; Diallo, Mamadou S; Simpson, André J; Liu, Yi; Goddard, William A; Kumar, Rajeev; Woods, Gwen C

2011-05-24

The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

DNA-binding study of anticancer drug cytarabine by spectroscopic and molecular docking techniques.

PubMed

Shahabadi, Nahid; Falsafi, Monireh; Maghsudi, Maryam

2017-01-02

The interaction of anticancer drug cytarabine with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multispectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove-binding mode, while the binding constant of UV-vis and the number of binding sites were 4.0 ± 0.2 × 10 4 L mol -1 and 1.39, respectively. The fluorimetric studies showed that the reaction between the drugs with CT-DNA is exothermic. Circular dichroism spectroscopy was employed to measure the conformational change of DNA in the presence of cytarabine. Furthermore, the drug induces detectable changes in its viscosity for DNA interaction. The molecular modeling results illustrated that cytarabine strongly binds to groove of DNA by relative binding energy of docked structure -20.61 KJ mol -1 . This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the interaction of small molecular pollutants and drugs with biomacromolecules for clarifying the molecular mechanism of toxicity or side effect in vivo.

Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

NASA Astrophysics Data System (ADS)

Shu, Chang; Ding, Li; Zhong, Wenying

2014-10-01

In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

PubMed

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-15

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-01

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques.

PubMed

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R; Abyar, Fatemeh

2018-06-05

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H 2 O] (1) and [Zn (L)] (2) [L=N-2-hydroxyacetophenon-N'-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1 H and 13 C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

2018-06-01

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

PubMed Central

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K.; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P.; Abell, Benjamin M.; Nabok, Alexei

2011-01-01

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. PMID:21767504

Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

NASA Astrophysics Data System (ADS)

Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

2016-04-01

In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus

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

Meisch, H.U.; Schmitt, J.A.

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of K/sub D/ = 1.59 x 10 M (pK/sub D/ = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals.more » From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed.« less

Characterization of interaction between doxycycline and human serum albumin by capillary electrophoresis-frontal analysis.

PubMed

Sun, Hanwen; He, Pan

2009-06-01

The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate, I=0.17, drug concentration 100 microM, HSA concentration up to 475 microM, 36.5 degrees C) was studied by CE-frontal analysis. The number of primary binding sites, binding constant and physiological protein-binding percentage were 1.9, 1.51 x 10(3) M(-1) and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (DeltaH), entropy change (DeltaS) and free energy change (DeltaG) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline-HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA.

Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin.

PubMed

Perry, Jennifer L; Goldsmith, Michael R; Williams, T Richard; Radack, Kyle P; Christensen, Trine; Gorham, Justin; Pasquinelli, Melissa A; Toone, Eric J; Beratan, David N; Simon, John D

2006-01-01

Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W-) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 x 10(5) M(-1). ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W- is accompanied by uptake of 0.30+/-0.02 protons from the solvent. This measurement suggests that the binding of W- is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W-.

Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

PubMed

Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

2014-05-01

The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

Theory for rates, equilibrium constants, and Brønsted slopes in F1-ATPase single molecule imaging experiments

PubMed Central

Volkán-Kacsó, Sándor; Marcus, Rudolph A.

2015-01-01

A theoretical model of elastically coupled reactions is proposed for single molecule imaging and rotor manipulation experiments on F1-ATPase. Stalling experiments are considered in which rates of individual ligand binding, ligand release, and chemical reaction steps have an exponential dependence on rotor angle. These data are treated in terms of the effect of thermodynamic driving forces on reaction rates, and lead to equations relating rate constants and free energies to the stalling angle. These relations, in turn, are modeled using a formalism originally developed to treat electron and other transfer reactions. During stalling the free energy profile of the enzymatic steps is altered by a work term due to elastic structural twisting. Using biochemical and single molecule data, the dependence of the rate constant and equilibrium constant on the stall angle, as well as the Børnsted slope are predicted and compared with experiment. Reasonable agreement is found with stalling experiments for ATP and GTP binding. The model can be applied to other torque-generating steps of reversible ligand binding, such as ADP and Pi release, when sufficient data become available. PMID:26483483

Spectroscopic and microcalorimetric studies on the molecular binding of food colorant acid red 27 with deoxyribonucleic acid.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2016-08-01

Interaction of the food colorant acid red 27 with double stranded DNA was investigated using spectroscopic and calorimetric methods. Absorbance and fluorescence studies suggested an intimate binding interaction between the dye and DNA. The quantum efficiency value testified an effective energy transfer from the DNA base pairs to the dye molecules. Minor groove displacement assay with Hoechst 33258 revealed that the binding occurs in the minor groove of DNA. Circular dichroism studies revealed that acid red 27 induces moderate conformational perturbations in DNA. Results of calorimetric studies suggested that the complexation process was driven largely by positive entropic contribution with a smaller favorable enthalpy contribution. The equilibrium constant of the binding was calculated to be (3.04 ± 0.09) × 10(4)  M(-1) at 298.15 K. Negative heat capacity value along with the enthalpy-entropy compensation phenomenon established the involvement of dominant hydrophobic forces in the binding process. Differential scanning calorimetry studies presented evidence for an increased thermal stability of DNA on binding of acid red 27. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis, characterization, redox behavior, DNA and protein binding and antibacterial activity studies of ruthenium(II) complexes of bidentate schiff bases.

PubMed

Paul, Hena; Sen, Buddhadeb; Mondal, Tapan Kumar; Chattopadhyay, Pabitra

2017-08-03

Two new ruthenium(II) complexes of Schiff base ligands (L) derived from cinnamaldehyde and ethylenediamine formulated as [Ru(L)(bpy) 2 ](ClO 4 ) 2 , where L 1 = N,N'-bis(4-nitrocinnamald-ehyde)ethylenediamine and L 2 = N,N'-bis(2-nitrocinnamaldehyde)-ethylenediamine for complex 1 and 2, respectively, were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The interaction of the complexes with calf thymus DNA (CT-DNA) using absorption, emission spectral studies and electrochemical techniques have been used to determine the binding constant, K b and the linear Stern-Volmer quenching constant, K SV . The results indicate that the ruthenium(II) complexes interact with CT-DNA strongly in a groove binding mode. The interactions of bovine serum albumin (BSA) with the complexes were also investigated with the help of absorption and fluorescence spectroscopy tools. Absorption spectroscopy proved the formation of a ground state BSA-[Ru(L)(bpy) 2 ](ClO 4 ) 2 complex. The antibacterial study showed that the Ru(II) complexes (1 and 2) have better activity than the standard antibiotics but weak activity than the ligands.

Kinetics of binding of chicken cystatin to papain.

PubMed

Björk, I; Alriksson, E; Ylinenjärvi, K

1989-02-21

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Host-guest chemistry of dendrimer-drug complexes. 4. An in-depth look into the binding/encapsulation of guanosine monophosphate by dendrimers.

PubMed

Hu, Jingjing; Fang, Min; Cheng, Yiyun; Zhang, Jiahai; Wu, Qinglin; Xu, Tongwen

2010-06-03

In the present study, we investigated the host-guest chemistry of dendrimer/guanosine monophosphate (GMP) and present an in-depth look into the binding/encapsulation of GMP by dendrimers using NMR studies. (1)H NMR spectra showed a significant downfield shift of methylene protons in the outmost layer of the G5 dendrimer, indicating the formation of ion pairs between cationic amine groups of dendrimer and anionic phosphate groups of GMP. Chemical shift titration results showed that the binding constant between G5 dendrimer and GMP is 17,400 M(-1) and each G5 dendrimer has 107 binding sites. The binding of GMP to dendrimers prevents its aggregation in aqueous solutions and thereby enhances its stability. Nuclear Overhauser effect measurements indicated that a GMP binding and encapsulation balance occurs on the surface and in the interior of dendrimer. The binding/encapsulation transitions can be easily tailored by altering the surface and interior charge densities of the dendrimer. All these findings provide a new insight into the host-guest chemistry of dendrimer/guest complexes and may play important roles in the study of dendrimer/DNA aggregates by a "bottom-up" strategy.

Constants for mercury binding by organic matter isolates from the Florida Everglades

USGS Publications Warehouse

Benoit, J.M.; Mason, R.P.; Gilmour, C.C.; Aiken, G.R.

2001-01-01

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand approach to estimate conditional stability constants for Hg complexes with DOM isolates collected from Florida Everglades surface waters. The isolates examined were the hydrophobic fraction of DOM from a eutrophic, sulfidic site (F1-HPoA) and the hydrophilic fraction from an oligotrophic, low-sulfide site (2BS-HPiA). Our experimental determinations utilized overall octanol-water partitioning coefficients (Dow) for 203Hg at 0.01 M chloride and across pH and DOM concentration gradients. Use of this radioisotope allowed rapid determinations of Hg concentrations in both water and octanol phases without problems of matrix interference. Conditional stability constants (1 = 0.06, 23??C) were log K??? = 11.8 for F1-HPoA and log K' = 10.6 for 2BS-HPiA. These are similar to previously published stability constants for Hg binding to low-molecular-weight thiols. Further, F1-HPoA showed a pH-dependent decline in Dow that was consistent with models of Hg complexation with thiol groups as the dominant Hg binding sites in DOM. These experiments demonstrate that the DOM isolates are stronger ligands for Hg than chloride ion or ethylenediamine-tetraacetic acid. Speciation calculations indicate that at the DOM concentrations frequently measured in Everglades, 20 to 40 ??M, significant complexation of Hg by DOM would be expected in aerobic (sulfide-free) surface waters. Copyright ?? 2001 Elsevier Science Ltd.

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8.

PubMed

Eron, Scott J; MacPherson, Derek J; Dagbay, Kevin B; Hardy, Jeanne A

2018-05-18

Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importers and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. In this work, we have adopted a method of assessing available zinc. This allowed assessment of accurate inhibition constants for apoptotic caspases, caspase-3, -6, -7, and -8. Each of these caspases are inhibited by zinc at intracellular levels but with widely differing inhibition constants and different zinc binding stoichiometries. Caspase-3, -6, and -8 appear to be constitutively inhibited by typical zinc levels, and this inhibition must be lifted to allow activation. The inhibition constant for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concentrations but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs, respectively. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a critical role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

PubMed

Athwal, Navjot Singh; Alagurajan, Jagannathan; Andreotti, Amy H; Hargrove, Mark S

2016-10-18

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

A Comparative Study on the Photophysics and Photochemistry of Xanthene Dyes in the Presence of Polyamidoamine (PAMAM) Dendrimers.

PubMed

Arbeloa, Ernesto Maximiliano; Previtali, Carlos Mario; Bertolotti, Sonia Graciela

2018-04-17

The photophysical and photochemical properties of the xanthene dyes Eosin Y, Erythrosin B, and Rose Bengal are evaluated in the presence of amino-terminated polyamidoamine (PAMAM) dendrimers of relatively high generation (G3-G5) in alkaline aqueous solution. UV/Vis absorption and fluorescence spectra of the dyes show bathochromic shifts, which correlate with the size of the dendrimer. Binding constants (K bind ) are calculated from absorption data. The resulting high K bind values indicate strong interactions between both molecules. Triplet-triplet absorption spectra of the dyes are recorded by laser flash photolysis, and a decrease in the triplet lifetimes is observed in the presence of dendrimers. At the same time, an increase in the absorption of the semireduced form of the dyes is observed. Rate constants for triplet quenching ( 3 k q ) and radical quantum yields (Φ R ) are obtained. The results are explained by a very efficient electron-transfer process from PAMAM to xanthene dyes for all of the dye/dendrimer couples that are evaluated. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrostatic correlations at the Stern layer: Physics or chemistry?

NASA Astrophysics Data System (ADS)

Travesset, A.; Vangaveti, S.

2009-11-01

We introduce a minimal free energy describing the interaction of charged groups and counterions including both classical electrostatic and specific interactions. The predictions of the model are compared against the standard model for describing ions next to charged interfaces, consisting of Poisson-Boltzmann theory with additional constants describing ion binding, which are specific to the counterion and the interfacial charge ("chemical binding"). It is shown that the "chemical" model can be appropriately described by an underlying "physical" model over several decades in concentration, but the extracted binding constants are not uniquely defined, as they differ depending on the particular observable quantity being studied. It is also shown that electrostatic correlations for divalent (or higher valence) ions enhance the surface charge by increasing deprotonation, an effect not properly accounted within chemical models. The charged phospholipid phosphatidylserine is analyzed as a concrete example with good agreement with experimental results. We conclude with a detailed discussion on the limitations of chemical or physical models for describing the rich phenomenology of charged interfaces in aqueous media and its relevance to different systems with a particular emphasis on phospholipids.

Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

PubMed Central

Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G.; Rummel, Andreas

2016-01-01

Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

Mass spectrometry reveals that the antibiotic simocyclinone D8 binds to DNA gyrase in a "bent-over" conformation: evidence of positive cooperativity in binding.

PubMed

Edwards, Marcus J; Williams, Mark A; Maxwell, Anthony; McKay, Adam R

2011-05-03

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.

Computational scheme for pH-dependent binding free energy calculation with explicit solvent.

PubMed

Lee, Juyong; Miller, Benjamin T; Brooks, Bernard R

2016-01-01

We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it. To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal mol(-1) . We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. © 2015 The Protein Society.

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

PubMed Central

Pal, Sandip; Dey, Subrata Kumar; Saha, Chabita

2014-01-01

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition. PMID:25025898

Spectroscopic characterisation of interaction of ferulic acid with aldehyde dehydrogenase (ALDH).

PubMed

Kolawole, Ayodele O; Agaba, Ruth J; Oluwole, Matthew O

2017-05-01

Interaction of a pharmacological important phenolic, ferulic acid, with Aldehyde dehydrogenase (ALDH) at the simulative pH condition, was studied using spectroscopic approach. Ferulic acid caused a decrease in the fluorescence intensity formed from ALDH-ferulic acid complex resulting in mixed inhibition of ALDH activity (IC 50 =30.65μM). The intrinsic quenching was dynamic and induced altered conformation of ALDH and made the protein less compact but might not unfold it. ALDH has two binding sites for ferulic acid at saturating concentrations having association constant of 1.35×10 3 Lmol -1 and a dissociation constant of 9.7×10 7 Lmol -1 at 25°C indicating ALDH-ferulic acid complex formation is more favourable than its dissociation. The interaction was not spontaneous and endothermic and suggests the involvement of hydrophobic interactions with a FRET binding distance of 4.49nm. Change in pH near and far from isoelectric points of ferulic acid did not affect the bonding interaction. Using trehalose as viscosogen, the result from Stoke-Einstein hypothesis showed that ferulic acid-ALDH binding and dissociation equilibrium was diffusion controlled. These results clearly suggest the unique binding properties and lipophilicity influence of ferulic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

Implications of effluent organic matter and its hydrophilic fraction on zinc(II) complexation in rivers under strong urban pressure: aromaticity as an inaccurate indicator of DOM-metal binding.

PubMed

Louis, Yoann; Pernet-Coudrier, Benoît; Varrault, Gilles

2014-08-15

The zinc binding characteristics of dissolved organic matter (DOM) fractions from the Seine River Basin were studied after being separated and extracted according to their polarity: hydrophobic, transphilic, and hydrophilic. The applied experimental methodology was based on a determination of labile zinc species by means of differential pulse anodic stripping voltammetry (DPASV) at increasing concentrations of total zinc on a logarithmic scale and at fixed levels of: pH, ionic strength, and temperature. Fitting the DOM fractions with two discrete classes of ligands successfully allowed determining the conditional zinc binding constants (Ki) as well as total ligand density (LiT). The binding constants obtained for each DOM fraction were then compared and discussed with respect to the hydrophobic/hydrophilic nature and sample origin. Results highlighted a strong complexation of zinc to the effluent organic matter and especially the most hydrophilic fraction, which also displayed a very low specific UV absorbance. Although the biotic ligand model takes into account the quality of DOM through UV absorbance in the predictions of metal bioavailability and toxicity, this correction is not efficient for urban waters. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca2+-ATPase: kinetic pathway and effects of methionine oxidation

PubMed Central

Slaughter, Brian D.; Bieber Urbauer, Ramona J.; Urbauer, Jeffrey L.; Johnson, Carey K.

2008-01-01

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca2+ pump. PMID:17343368

Interactions of tea tannins and condensed tannins with proteins.

PubMed

Frazier, Richard A; Deaville, Eddie R; Green, Rebecca J; Stringano, Elisabetta; Willoughby, Ian; Plant, John; Mueller-Harvey, Irene

2010-01-20

Binding parameters for the interactions of four types of tannins: tea catechins, grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins, and sorghum procyanidins (mDP=17), with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction with gelatin were in the range 10(4) to 10(6) M(-1) and in the order: sorghum procyanidins > grape seed proanthocyanidins > mimosa 5-deoxy proanthocyanidins > tea catechins. Interaction with BSA was generally weaker, with equilibrium binding constants of < or =10(3)M(-1) for grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins and tea catechins, and 10(4)M(-1) for the sorghum procyanidins. In all cases the interactions with proteins were exothermic and involved multiple binding sites on the protein. The data are discussed in relation to the structures and the known nutritional effects of the condensed tannins.

Photocaged Competitor Guests: A General Approach Toward Light-Activated Cargo Release From Cucurbiturils.

PubMed

Romero, Miguel A; Basílio, Nuno; Moro, Artur J; Domingues, Mara; González-Delgado, José A; Arteaga, Jesús F; Pischel, Uwe

2017-09-21

A general approach toward the light-induced guest release from cucurbit[7]uril by means of a photoactivatable competitor was devised. An o-nitrobenzyl-caged competitor is photolyzed to generate a competitive guest that can displace cargo from the host macrocycle solely based on considerations of chemical equilibrium. With this method the release of terpene guests from inclusion complexes with cucurbit[7]uril was demonstrated. The binding of the herein investigated terpenes, all being lead fragrant components in essential oils, has been characterized for the first time. They feature binding constants of up to 10 8  L mol -1 and a high differential binding selectivity (spanning four orders of magnitude for the binding constants for the particular set of terpenes). By fine-tuning the photoactivatable competitor guest, selective and also sequential release of the terpenes was achieved. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

PubMed

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

PubMed

Hoggett, J G; Kellett, G L

1976-06-15

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.

Interaction between Pin1 and its natural product inhibitor epigallocatechin-3-gallate by spectroscopy and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Xi, Lei; Wang, Yu; He, Qing; Zhang, Qingyan; Du, Linfang

2016-12-01

The binding of epigallocatechin-3-gallate (EGCG) to wild type Pin1 in solution was studied by spectroscopic methods and molecular dynamics simulations in this research to explore the binding mode and inhibition mechanism. The binding constants and number of binding sites per Pin1 for EGCG were calculated through the Stern-Volmer equation. The values of binding free energy and thermodynamic parameters were calculated and indicated that hydrogen bonds, electrostatic interaction and Van der Waals interaction played the major role in the binding process. The alterations of Pin1 secondary structure in the presence of EGCG were confirmed by far-UV circular dichroism spectra. The binding model at atomic-level revealed that EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain. Furthermore, EGCG could also interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domain.

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

PubMed Central

Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

Using two-dimensional correlation size exclusion chromatography (2D-CoSEC) to explore the size-dependent heterogeneity of humic substances for copper binding.

PubMed

Lee, Yun-Kyung; Hur, Jin

2017-08-01

Knowledge of the heterogeneous distribution of humic substances (HS) reactivities along a continuum of molecular weight (MW) is crucial for the systems where the HS MW is subject to change. In this study, two dimensional correlation spectroscopy combined with size exclusion chromatography (2D-CoSEC) was first utilized to obtain a continuous and heterogeneous presence of copper binding characteristics within bulk HS with respect to MW. HS solutions with varying copper concentrations were directly injected into a size exclusion chromatography (SEC) system with Tris-HCl buffer as a mobile phase. Several validation tests confirmed neither structural disruption of HS nor competition effect of the mobile phase used. Similar to batch systems, fluorescence quenching was observed in the chromatograms over a wide range of HS MW. 2D-CoSEC maps of a soil-derived HS (Elliot soil humic acid) showed the greater fluorescence quenching degrees with respect to the apparent MW on the order of 12500 Da > 10600 Da > 7000 Da > 15800 Da. The binding constants calculated based on modified Stern-Volmer equation were consistent with the 2D-CoSEC results. More heterogeneity of copper binding affinities within bulk HS was found for the soil-derived HS versus an aquatic HS. The traditional fluorescence quenching titration method using ultrafiltered HS size fractions failed to delineate detailed distribution of the copper binding characteristics, exhibiting a much shorter range of the binding constants than those obtained from the 2D-CoSEC. Our proposed technique demonstrated a great potential to describe metal binding characteristics of HS at high MW resolution, providing a clear picture of the size-dependent metal-HS interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the Organic Component of Low-Molecular-Weight Chromium-Binding Substance and Its Binding of Chromium123

PubMed Central

Chen, Yuan; Watson, Heather M.; Gao, Junjie; Sinha, Sarmistha Halder; Cassady, Carolyn J.; Vincent, John B.

2011-01-01

Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism. PMID:21593351

Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

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

Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using ({sup 125}I)melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37{degree}C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of ({sup 125}I)melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8more » fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of ({sup 125}I)melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the ({sup 125}I)melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.« less

Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

ERIC Educational Resources Information Center

Hicks, Katherine A.

2016-01-01

Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

Kinetic intermediates of unfolding of dimeric prostatic phosphatase.

PubMed

Kuciel, Radosława; Mazurkiewicz, Aleksandra; Dudzik, Paulina

2007-01-01

Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20 degrees C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 +/- 0.0024 min(-1) . The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 +/- 0.0018 min(-1) and 0.0409 +/- 0.0052 min(-1), respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 +/- 0.014 min(-1) and 0.216 +/- 0.010 min(-1), respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.

Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from Rhodobacter sphaeroides.

PubMed

Espiritu, Eduardo; Olson, Tien L; Williams, JoAnn C; Allen, James P

2017-12-12

The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to Q A , the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P + , with a dissociation constant of 1.2 μM at pH 9.4, comparable to the binding constant of c-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 μM. The extent of reduction of P + by the P1 Mn-protein was dependent on the P/P + midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of -56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation-reduction reactions.

Interactions of high-affinity cationic blockers with the translocation pores of B. anthracis, C. botulinum, and C. perfringens binary toxins.

PubMed

Bezrukov, Sergey M; Liu, Xian; Karginov, Vladimir A; Wein, Alexander N; Leppla, Stephen H; Popoff, Michel R; Barth, Holger; Nestorovich, Ekaterina M

2012-09-19

Cationic β-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of three binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component, which acts on certain intracellular targets, and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. Here we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different β-cyclodextrins (AmPrβCD and AMBnTβCD) in the PA(63), C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTβCD, demonstrated significantly stronger binding compared with AmPrβCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnTβCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrβCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration-independent, short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA(63), which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geologic settings

USGS Publications Warehouse

Borrok, D.; Turner, B.F.; Fein, J.B.

2005-01-01

Adsorption onto bacterial cell walls can significantly affect the speciation and mobility of aqueous metal cations in many geologic settings. However, a unified thermodynamic framework for describing bacterial adsorption reactions does not exist. This problem originates from the numerous approaches that have been chosen for modeling bacterial surface protonation reactions. In this study, we compile all currently available potentiometric titration datasets for individual bacterial species, bacterial consortia, and bacterial cell wall components. Using a consistent, four discrete site, non-electrostatic surface complexation model, we determine total functional group site densities for all suitable datasets, and present an averaged set of 'universal' thermodynamic proton binding and site density parameters for modeling bacterial adsorption reactions in geologic systems. Modeling results demonstrate that the total concentrations of proton-active functional group sites for the 36 bacterial species and consortia tested are remarkably similar, averaging 3.2 ?? 1.0 (1??) ?? 10-4 moles/wet gram. Examination of the uncertainties involved in the development of proton-binding modeling parameters suggests that ignoring factors such as bacterial species, ionic strength, temperature, and growth conditions introduces relatively small error compared to the unavoidable uncertainty associated with the determination of cell abundances in realistic geologic systems. Hence, we propose that reasonable estimates of the extent of bacterial cell wall deprotonation can be made using averaged thermodynamic modeling parameters from all of the experiments that are considered in this study, regardless of bacterial species used, ionic strength, temperature, or growth condition of the experiment. The average site densities for the four discrete sites are 1.1 ?? 0.7 ?? 10-4, 9.1 ?? 3.8 ?? 10-5, 5.3 ?? 2.1 ?? 10-5, and 6.6 ?? 3.0 ?? 10-5 moles/wet gram bacteria for the sites with pKa values of 3.1, 4.7, 6.6, and 9.0, respectively. It is our hope that this thermodynamic framework for modeling bacteria-proton binding reactions will also provide the basis for the development of an internally consistent set of bacteria-metal binding constants. 'Universal' constants for bacteria-metal binding reactions can then be used in conjunction with equilibrium constants for other important metal adsorption and complexation reactions to calculate the overall distribution of metals in realistic geologic systems.

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.

PubMed

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P; Abell, Benjamin M; Nabok, Alexei

2011-07-20

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

DNA binding of a proflavine derivative bearing a platinum hanging residue.

PubMed

Biagini, Silvia; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Nikolayenko, Igor V; Secco, Fernando; Venturini, Marcella

2011-04-01

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides. Copyright © 2010 Elsevier Inc. All rights reserved.

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Modeling Complex Equilibria in ITC Experiments: Thermodynamic Parameters Estimation for a Three Binding Site Model

PubMed Central

Le, Vu H.; Buscaglia, Robert; Chaires, Jonathan B.; Lewis, Edwin A.

2013-01-01

Isothermal Titration Calorimetry, ITC, is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g. Keq (or ΔG), ΔH, ΔS, and n) for a ligand binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combination of equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding need to be developed on a case by case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the non-linear regression analysis of a multiple binding site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g. up to nine parameters in the three binding site model) yields thermodynamic parameters with acceptable accuracy. PMID:23262283

Specific binding of large aggregates of amphiphilic molecules to the respective antibodies.

PubMed

Nabok, Alexei; Tsargorodskaya, Anna; Holloway, Alan; Starodub, Nikolay F; Demchenko, Anna

2007-07-31

The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.

RNA adducts with Na 2SeO 4 and Na 2SeO 3 - Stability and structural features

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Manouchehri, Firouzeh; Montazeri, Maryam

2011-12-01

Selenium compounds are widely available in dietary supplements and have been extensively studied for their antioxidant and anticancer properties. Low blood Se levels were found to be associated with an increased incidence and mortality from various types of cancers. Although many in vivo and clinical trials have been conducted using these compounds, their biochemical and chemical mechanisms of efficacy are the focus of much current research. This study was designed to examine the interaction of Na 2SeO 4 and Na 2SeO 3 with RNA in aqueous solution at physiological conditions, using a constant RNA concentration (6.25 mM) and various sodium selenate and sodium selenite/polynucleotide (phosphate) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. Fourier transform infrared, UV-Visible spectroscopic methods were used to determine the drug binding modes, the binding constants, and the stability of Na 2SeO 4 and Na 2SeO 3-RNA complexes in aqueous solution. Spectroscopic evidence showed that Na 2SeO 4 and Na 2SeO 3 bind to the major and minor grooves of RNA ( via G, A and U bases) with some degree of the Se-phosphate (PO 2) interaction for both compounds with overall binding constants of K(Na 2SeO 4-RNA) = 8.34 × 10 3 and K(Na 2SeO 3-RNA) = 4.57 × 10 3 M -1. The order of selenium salts-biopolymer stability was Na 2SeO 4-RNA > Na 2SeO 3-RNA. RNA aggregations occurred at higher selenium concentrations. No biopolymer conformational changes were observed upon Na 2SeO 4 and Na 2SeO 3 interactions, while RNA remains in the A-family structure.

Fluorescent analysis of interaction of flavonols with hemoglobin and bovine serum albumin

NASA Astrophysics Data System (ADS)

Sentchouk, V. V.; Bondaryuk, E. V.

2007-09-01

We have studied the fluorescent properties of flavonols (quercetin, fisetin, morin, rutin) with the aim of studying possible interaction with hemoglobin and bovine serum albumin (BSA). We observed an increase in the intensity of intrinsic fluorescence for all the flavonols except rutin in the presence of BSA. From the changes in the fluorescence spectra, we concluded that tautomeric forms are formed on interaction with hemoglobin. We determined the interconnection between the structure of related flavonols and their fluorescent properties on interaction with proteins, and we determined the binding constants for binding with BSA and hemoglobin.

Relative Propensities of Cytochrome c Oxidase and Cobalt Corrins for Reaction with Cyanide and Oxygen: Implications for Amelioration of Cyanide Toxicity.

PubMed

Yuan, Quan; Pearce, Linda L; Peterson, Jim

2017-12-18

In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, K f1 = 1.1 (±0.6) × 10 5 M -1 and K f2 = 8.5 (±0.1) × 10 4 M -1 , or an overall cyanide binding constant of ∼1 × 10 10 M -2 . In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 10 4 M -1 and 1.6 (±0.2) × 10 7 M -1 , respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 10 3 M -1 s -1 and 45 (±1) M -1 s -1 , respectively. Under the same conditions, cobalamin exhibited a single slow cyanide-binding kinetic phase with a second-order rate constant of 35 (±1) M -1 s -1 . All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>10 6 M -1 s -1 ). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent reactions could potentially lead to erroneous assessment of the ability of the cyanide scavenger to restore the activity of cyanide-inhibited cytochrome c oxidase.

Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

PubMed Central

2015-01-01

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs. PMID:25226846

Comparison of Calculation and Experiment Implicates Significant Electrostatic Contributions to the Binding Stability of Barnase and Barstar

PubMed Central

Dong, Feng; Vijayakumar, M.; Zhou, Huan-Xiang

2003-01-01

The contributions of electrostatic interactions to the binding stability of barnase and barstar were studied by the Poisson-Boltzmann model with three different protocols: a), the dielectric boundary specified as the van der Waals (vdW) surface of the protein along with a protein dielectric constant (ɛp) of 4; b), the dielectric boundary specified as the molecular (i.e., solvent-exclusion (SE)) surface along with ɛp = 4; and c), “SE + ɛp = 20.” The “vdW + ɛp = 4” and “SE + ɛp = 20” protocols predicted an overall electrostatic stabilization whereas the “SE + ɛp = 4” protocol predicted an overall electrostatic destabilization. The “vdW + ɛp = 4” protocol was most consistent with experiment. It quantitatively reproduced the observed effects of 17 mutations neutralizing charged residues lining the binding interface and the measured coupling energies of six charge pairs across the interface and reasonably rationalized the experimental ionic strength and pH dependences of the binding constant. In contrast, the “SE + ɛp = 4” protocol predicted significantly larger coupling energies of charge pairs whereas the “SE + ɛp = 20” protocol did not predict any pH dependence. This study calls for further scrutiny of the different Poisson-Boltzmann protocols and demonstrates potential danger in drawing conclusions on electrostatic contributions based on a particular calculation protocol. PMID:12829463

Sensing of Double-Stranded DNA/RNA Secondary Structures by Water Soluble Homochiral Perylene Bisimide Dyes.

PubMed

Gershberg, Jana; Radić Stojković, Marijana; Škugor, Marko; Tomić, Sanja; Rehm, Thomas H; Rehm, Stefanie; Saha-Möller, Chantu R; Piantanida, Ivo; Würthner, Frank

2015-05-18

A broad series of homochiral perylene bisimide (PBI) dyes were synthesized that are appended with amino acids and cationic side chains at the imide positions. Self-assembly behavior of these ionic PBIs has been studied in aqueous media by UV/Vis spectroscopy, revealing formation of excitonically coupled H-type aggregates. The interactions of these ionic PBIs with different ds-DNA and ds-RNA have been explored by thermal denaturation, fluorimetric titration and circular dichroism (CD) experiments. These PBIs strongly stabilized ds-DNA/RNA against thermal denaturation as revealed by high melting temperatures of the formed PBI/polynucleotide complexes. Fluorimetric titrations showed that these PBIs bind to ds-DNA/RNA with high binding constants depending on the number of the positive charges in the side chains. Thus, spermine-containing PBIs with six positive charges each showed higher binding constants (logKs =9.2-9.8) than their dioxa analogues (logKs =6.5-7.9) having two positive charges each. Induced circular dichroism (ICD) of PBI assemblies created within DNA/RNA grooves was observed. These ICD profiles are strongly dependent on the steric demand of the chiral substituents of the amino acid units and the secondary structure of the DNA or RNA. The observed ICD effects can be explained by non-covalent binding of excitonically coupled PBI dimer aggregates into the minor groove of DNA and major groove of RNA which is further supported by molecular modeling studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation on the interaction of Rutin with serum albumins: Insights from spectroscopic and molecular docking techniques.

PubMed

Sengupta, Priti; Sardar, Pinki Saha; Roy, Pritam; Dasgupta, Swagata; Bose, Adity

2018-06-01

The binding interaction of Rutin, a flavonoid, with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), were investigated using different spectroscopic techniques, such as fluorescence, time-resolved single photon counting (TCSPC) and circular dichroism (CD) spectroscopy as well as molecular docking method. The emission studies revealed that the fluorescence quenching of BSA/HSA by Rutin occurred through a simultaneous static and dynamic quenching process, and we have evaluated both the quenching constants individually. The binding constants of Rutin-BSA and Rutin-HSA system were found to be 2.14 × 10 6  M -1 and 2.36 × 10 6  M -1 at 298 K respectively, which were quite high. Further, influence of some biologically significant metal ions (Ca 2+ , Zn 2+ and Mg 2+ ) on binding of Rutin to BSA and HSA were also investigated. Thermodynamic parameters justified the involvement of hydrogen bonding and weak van der Waals forces in the interaction of Rutin with both BSA and HSA. Further a site-marker competitive experiment was performed to evaluate Rutin binding site in the albumins. Additionally, the CD spectra of BSA and HSA revealed that the secondary structure of the proteins was perturbed in the presence of Rutin. Finally protein-ligand docking studies have also been performed to determine the probable location of the ligand molecule. Copyright © 2018 Elsevier B.V. All rights reserved.

Ontogenesis of the angiotensin II (ANGII) receptor system in the duck brain.

PubMed

Müller, A R; Gerstberger, R

1994-03-18

The ontogenetic development of the central nervous angiotensin II (ANGII) receptor system in the duck was studied at embryonic days E20 and E27 and at postnatal days P3 and P14 by computerized semiquantitative autoradiography employing the receptor antagonist 125I[1Sar,8Ile]ANGII as radioligand. For circumventricular structures involved in the sensing of brain-intrinsic (AV3V region) or blood-borne (subfornical organ, SFO) ANGII, binding sites for 125I[1Sar,8Ile]ANGII were first detectable at E27, with a steady rise in binding density up to P14. The choroid plexus of the lateral (PCVL) and third (PCVIII) cerebral ventricles responsible for cerebrospinal fluid (CSF) production were endowed with maximal ANGII receptor densities at E20 with subsequent reduction to constant medium (PCVIII) or low (PCVL) values. Besides the choroid plexus, the magnocellular paraventricular nucleus (PVN) was the only structure presenting ANGII specific binding sites at E20, although at low density. As for the SFO and AV3V region, labelling of ANGII binding sites in the PVN increased continuously during development to high values at P14. Nuclear components of the limbic system (archistriatum, amygdala and habenular complex) did not reveal specific labelling by the radioligand at E20 with constant, moderate binding densities evaluated for E27, P3 and P14. In the duck brain, functionally related structures exhibited a homogeneous ontogenetic development of their ANGII receptor system.

Binding of cyclic carboxylates to octa-acid deep-cavity cavitand

NASA Astrophysics Data System (ADS)

Gibb, Corinne L. D.; Gibb, Bruce C.

2014-04-01

As part of the fourth statistical assessment of modeling of proteins and ligands (sampl.eyesopen.com) prediction challenge, the strength of association of nine guests ( 1- 9) binding to octa-acid host was determined by a combination of 1H NMR and isothermal titration calorimetry. Association constants in sodium tetraborate buffered (pH 9.2) aqueous solution ranged from 5.39 × 102 M-1 in the case of benzoate 1, up to 3.82 × 105 M-1 for trans-4-methylcyclohexanoate 7. Overall, the free energy difference between the free energies of complexation of these weakest and strongest binding guests was ΔΔG° = 3.88 kcal mol-1. Based on a multitude of previous studies, the anticipated order of strength of binding was close to that which was actually obtained. However, the binding of guest 3 (4-ethylbenzoate) was considerably stronger than initially estimated.

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

2012-07-01

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

Determination of the binding properties of p-cresyl glucuronide to human serum albumin.

PubMed

Yi, Dan; Monteiro, Elisa Bernardes; Chambert, Stéphane; Soula, Hédi A; Daleprane, Julio B; Soulage, Christophe O

2018-04-26

p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×10 3  M -1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin. Copyright © 2018. Published by Elsevier B.V.

Mononuclear Pd(II) complex as a new therapeutic agent: Synthesis, characterization, biological activity, spectral and DNA binding approaches

NASA Astrophysics Data System (ADS)

Saeidifar, Maryam; Mirzaei, Hamidreza; Ahmadi Nasab, Navid; Mansouri-Torshizi, Hassan

2017-11-01

The binding ability between a new water-soluble palladium(II) complex [Pd(bpy)(bez-dtc)]Cl (where bpy is 2,2‧-bipyridine and bez-dtc is benzyl dithiocarbamate), as an antitumor agent, and calf thymus DNA was evaluated using various physicochemical methods, such as UV-Vis absorption, Competitive fluorescence studies, viscosity measurement, zeta potential and circular dichroism (CD) spectroscopy. The Pd(II) complex was synthesized and characterized using elemental analysis, molar conductivity measurements, FT-IR, 1H NMR, 13C NMR and electronic spectra studies. The anticancer activity against HeLa cell lines demonstrated lower cytotoxicity than cisplatin. The binding constants and the thermodynamic parameters were determined at different temperatures (300 K, 310 K and 320 K) and shown that the complex can bind to DNA via electrostatic forces. Furthermore, this result was confirmed by the viscosity and zeta potential measurements. The CD spectral results demonstrated that the binding of Pd(II) complex to DNA induced conformational changes in DNA. We hope that these results will provide a basis for further studies and practical clinical use of anticancer drugs.

Effect of temperature on the methotrexate BSA interaction: Spectroscopic study

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Maciążek, M.; Równicka, J.; Bojko, B.; Pentak, D.; Sułkowski, W. W.

2007-05-01

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory illness which affects about one percent of the world's population. Methotrexate (4-amino-10-methylfolic acid) (MTX) also known as amethopterin is commonly used to treat rheumatoid arthritis (RA). It is transported in the circulary system as a complex with serum albumin. The aim of this study was to investigate the interactions of MTX with transporting protein with the use of spectroscopic methods. The binding of MTX to bovine serum albumin (BSA) was studied by monitoring the changes in the emission fluorescence spectra of protein in the presence of MTX at excitation wavelength of 280 nm and 295 nm. The quenching of protein fluorescence at temperature range from 298 K to 316 K was observed. Energy transfer between methotrexate and fluorophores contained in the serum albumin structure was found at the molar ratio MTX:BSA 7.5:1. The relative fluorescence intensity of BSA decreases with increase of temperature. Similar results were observed for BSA excited with 280 nm and 295 nm at the same temperature range. The presence of MTX seems to prevent these changes. Temperature dependence of the binding constant has been presented. The binding and quenching constants for equilibrium complex were calculated using Scatchard and Stern-Volmer method, respectively. The results show that MTX forms π-π complex with aromatic amino acid residues of BSA. The binding site for MTX on BSA was found to be situated in the hydrophobic IIA or IB subdomain where the Trps were located. The spontaneity of MTX-BSA complex formation in the temperature range 298-316 K was ascertained.

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

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

Morrison, W.J.; Offner, H.; Vandenbark, A.A.

1989-01-01

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete†

PubMed Central

Tjepkema, John D.; Cashon, Robert E.; Beckwith, Jason; Schwintzer, Christa R.

2002-01-01

Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1. PMID:11976149

Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

PubMed

Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

2012-06-04

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supramolecular complex of a fused zinc phthalocyanine-zinc porphyrin dyad assembled by two imidazole-C60 units: ultrafast photoevents.

PubMed

Follana-Berná, Jorge; Seetharaman, Sairaman; Martín-Gomis, Luis; Charalambidis, Georgios; Trapali, Adelais; Karr, Paul A; Coutsolelos, Athanassios G; Fernández-Lázaro, Fernando; D'Souza, Francis; Sastre-Santos, Ángela

2018-03-14

A new zinc phthalocyanine-zinc porphyrin dyad (ZnPc-ZnP) fused through a pyrazine ring has been synthesized as a receptor for imidazole-substituted C 60 (C 60 Im) electron acceptor. Self-assembly via metal-ligand axial coordination and the pertinent association constants in solution were determined by 1 H-NMR, UV-Vis and fluorescence titration experiments at room temperature. The designed host was able to bind up to two C 60 Im electron acceptor guest molecules to yield C 60 Im:ZnPc-ZnP:ImC 60 donor-acceptor supramolecular complex. The spectral data showed that the two binding sites behave independently with binding constants similar in magnitude. Steady-state fluorescence studies were indicative of an efficient singlet-singlet energy transfer from zinc porphyrin to zinc phthalocyanine within the fused dyad. Accordingly, the transient absorption studies covering a wide timescale of femto-to-milli seconds revealed ultrafast energy transfer from 1 ZnP* to ZnPc (k EnT ∼ 10 12 s -1 ) in the fused dyad. Further, a photo induced electron transfer was observed in the supramolecularly assembled C 60 Im:ZnPc-ZnP:ImC 60 donor-acceptor complex leading to charge separated states, which persisted for about 200 ns.

Trace Metal-Humic Complexes in Natural Waters: Insights From Speciation Experiments

NASA Astrophysics Data System (ADS)

Stern, J. C.; Salters, V.; Sonke, J.

2006-12-01

The DOM cycle is intimately linked to the cycling and bioavailability of trace metals in aqueous environments. The presence or absence of DOM in the water column can determined whether trace elements will be present in limited quantities as a nutrient, or in surplus quantities as a toxicant. Humic substances (HS), which represent the refractory products of DOM degradation, strongly affect the speciation of trace metals in natural waters. To simulate metal-HS interactions in nature, experiments must be carried out using trace metal concentrations. Sensitive detection systems such as ICP-MS make working with small (nanomolar) concentrations possible. Capillary electrophoresis coupled with ICP-MS (CE-ICP-MS) has recently been identified as a rapid and accurate method to separate metal species and calculate conditional binding constants (log K_c) of metal-humic complexes. CE-ICP-MS was used to measure partitioning of metals between humic substances and a competing ligand (EDTA) and calculate binding constants of rare earth element (REE) and Th, Hf, and Zr-humic complexes at pH 3.5-8 and ionic strength of 0.1. Equilibrium dialysis ligand exchange (EDLE) experiments to validate the CE-ICP-MS method were performed to separate the metal-HS and metal-EDTA species by partitioning due to size exclusion via diffusion through a 1000 Da membrane. CE-ICP-MS experiments were also conducted to compare binding constants of REE with humic substances of various origin, including soil, peat, and aquatic DOM. Results of our experiments show an increase in log K_c with decrease in ionic radius for REE-humic complexes (the lanthanide contraction effect). Conditional binding constants of tetravalent metal-humic complexes were found to be several orders of magnitude higher than REE-humic complexes, indicating that tetravalent metals have a very strong affinity for humic substances. Because thorium is often used as a proxy for the tetravalent actinides, Th-HS binding constants can allow us to assess the importance of tetravalent actinide-humic complexes in groundwater transport from nuclear repositories. Our results suggest that tetravalent actinide-humic complexes couild be more important to account for in predictive speciation models than previously thought.

An artificial guanine that binds cytidine through the cooperative interaction of metal coordination and hydrogen bonding.

PubMed

Mancin, Fabrizio; Chin, Jik

2002-09-18

Cd(II) complex of L binds selectively to cytidine in DMSO with an equilibrium constant of 117 M-1 (where LH is 2-aminomethyl-8-hydroxyquinoline). In contrast, the Zn(II) complex of L does not bind appreciably to any of the four nucleobases under the same condition used for the Cd(II) complex.

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin.

PubMed

Choudhury, Diptiman; Ganguli, Arnab; Dastidar, Debabrata Ghosh; Acharya, Bipul R; Das, Amlan; Chakrabarti, Gopal

2013-06-01

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The role of multivalency in the association kinetics of patchy particle complexes.

PubMed

Newton, Arthur C; Groenewold, Jan; Kegel, Willem K; Bolhuis, Peter G

2017-06-21

Association and dissociation of particles are elementary steps in many natural and technological relevant processes. For many such processes, the presence of multiple binding sites is essential. For instance, protein complexes and regular structures such as virus shells are formed from elementary building blocks with multiple binding sites. Here we address a fundamental question concerning the role of multivalency of binding sites in the association kinetics of such complexes. Using single replica transition interface sampling simulations, we investigate the influence of the multivalency on the binding kinetics and the association mechanism of patchy particles that form polyhedral clusters. When the individual bond strength is fixed, the kinetics naturally is very dependent on the multivalency, with dissociation rate constants exponentially decreasing with the number of bonds. In contrast, we find that when the total bond energy per particle is kept constant, association and dissociation rate constants turn out rather independent of multivalency, although of course still very dependent on the total energy. The association and dissociation mechanisms, however, depend on the presence and nature of the intermediate states. For instance, pathways that visit intermediate states are less prevalent for particles with five binding sites compared to the case of particles with only three bonds. The presence of intermediate states can lead to kinetic trapping and malformed aggregates. We discuss implications for natural forming complexes such as virus shells and for the design of artificial colloidal patchy particles.

The role of multivalency in the association kinetics of patchy particle complexes

NASA Astrophysics Data System (ADS)

Newton, Arthur C.; Groenewold, Jan; Kegel, Willem K.; Bolhuis, Peter G.

2017-06-01

Association and dissociation of particles are elementary steps in many natural and technological relevant processes. For many such processes, the presence of multiple binding sites is essential. For instance, protein complexes and regular structures such as virus shells are formed from elementary building blocks with multiple binding sites. Here we address a fundamental question concerning the role of multivalency of binding sites in the association kinetics of such complexes. Using single replica transition interface sampling simulations, we investigate the influence of the multivalency on the binding kinetics and the association mechanism of patchy particles that form polyhedral clusters. When the individual bond strength is fixed, the kinetics naturally is very dependent on the multivalency, with dissociation rate constants exponentially decreasing with the number of bonds. In contrast, we find that when the total bond energy per particle is kept constant, association and dissociation rate constants turn out rather independent of multivalency, although of course still very dependent on the total energy. The association and dissociation mechanisms, however, depend on the presence and nature of the intermediate states. For instance, pathways that visit intermediate states are less prevalent for particles with five binding sites compared to the case of particles with only three bonds. The presence of intermediate states can lead to kinetic trapping and malformed aggregates. We discuss implications for natural forming complexes such as virus shells and for the design of artificial colloidal patchy particles.

Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Santos, Inês C.; Waybright, Veronica B.; Fan, Hui; Ramirez, Sabra; Mesquita, Raquel B. R.; Rangel, António O. S. S.; Fryčák, Petr; Schug, Kevin A.

2015-07-01

Described is a new method based on the concept of controlled band dispersion, achieved by hyphenating flow injection analysis with ESI-MS for noncovalent binding determinations. A continuous stirred tank reactor (CSTR) was used as a FIA device for exponential dilution of an equimolar host-guest solution over time. The data obtained was treated for the noncovalent binding determination using an equimolar binding model. Dissociation constants between vancomycin and Ac-Lys(Ac)-Ala-Ala-OH peptide stereoisomers were determined using both the positive and negative ionization modes. The results obtained for Ac- L-Lys(Ac)- D-Ala- D-Ala (a model for a Gram-positive bacterial cell wall) binding were in reasonable agreement with literature values made by other mass spectrometry binding determination techniques. Also, the developed method allowed the determination of dissociation constants for vancomycin with Ac- L-Lys(Ac)- D-Ala- L-Ala, Ac- L-Lys(Ac)- L-Ala- D-Ala, and Ac- L-Lys(Ac)- L-Ala- L-Ala. Although some differences in measured binding affinities were noted using different ionization modes, the results of each determination were generally consistent. Differences are likely attributable to the influence of a pseudo-physiological ammonium acetate buffer solution on the formation of positively- and negatively-charged ionic complexes.

A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants.

PubMed

Drake, Andrew W; Klakamp, Scott L

2007-01-10

A new 4-parameter nonlinear equation based on the standard multiple independent binding site model (MIBS) is presented for fitting cell-based ligand titration data in order to calculate the ligand/cell receptor equilibrium dissociation constant and the number of receptors/cell. The most commonly used linear (Scatchard Plot) or nonlinear 2-parameter model (a single binding site model found in commercial programs like Prism(R)) used for analysis of ligand/receptor binding data assumes only the K(D) influences the shape of the titration curve. We demonstrate using simulated data sets that, depending upon the cell surface receptor expression level, the number of cells titrated, and the magnitude of the K(D) being measured, this assumption of always being under K(D)-controlled conditions can be erroneous and can lead to unreliable estimates for the binding parameters. We also compare and contrast the fitting of simulated data sets to the commonly used cell-based binding equation versus our more rigorous 4-parameter nonlinear MIBS model. It is shown through these simulations that the new 4-parameter MIBS model, when used for cell-based titrations under optimal conditions, yields highly accurate estimates of all binding parameters and hence should be the preferred model to fit cell-based experimental nonlinear titration data.

High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

PubMed Central

Wittenberg, Nathan J.; Im, Hyungsoon; Xu, Xiaohua; Wootla, Bharath; Watzlawik, Jens; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

2012-01-01

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (KD ~0.9 nM) for natural autoantibodies. This is approximately one to two orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair. PMID:22762372

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB*

PubMed Central

Creager-Allen, Rachel L.; Silversmith, Ruth E.; Bourret, Robert B.

2013-01-01

Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3−. Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ∼10-fold higher than for the monomer. In a test of the model, disruption of the known PhoBN dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation. PMID:23760278

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum.

PubMed

Dixit, Ajay Kumar; Jayabaskaran, Chelliah

2015-05-01

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca(2+) ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 μM and 120 μM indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 μM and 1.7 μM. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a α-helical rich protein. Calcium binding further increased the α-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. Copyright © 2015 Elsevier GmbH. All rights reserved.

An Ultraviolet Resonance Raman Spectroscopic Study of Cisplatin and Transplatin Interactions with Genomic DNA.

PubMed

Geng, Jiafeng; Aioub, Mena; El-Sayed, Mostafa A; Barry, Bridgette A

2017-09-28

Ultraviolet resonance Raman (UVRR) spectroscopy is a label-free method to define biomacromolecular interactions with anticancer compounds. Using UVRR, we describe the binding interactions of two Pt(II) compounds, cisplatin (cis-diamminedichloroplatinum(II)) and its isomer, transplatin, with nucleotides and genomic DNA. Cisplatin binds to DNA and other cellular components and triggers apoptosis, whereas transplatin is clinically ineffective. Here, a 244 nm UVRR study shows that purine UVRR bands are altered in frequency and intensity when mononucleotides are treated with cisplatin. This result is consistent with previous suggestions that purine N7 provides the cisplatin-binding site. The addition of cisplatin to DNA also causes changes in the UVRR spectrum, consistent with binding of platinum to purine N7 and disruption of hydrogen-bonding interactions between base pairs. Equally important is that transplatin treatment of DNA generates similar UVRR spectral changes, when compared to cisplatin-treated samples. Kinetic analysis, performed by monitoring decreases of the 1492 cm -1 band, reveals biphasic kinetics and is consistent with a two-step binding mechanism for both platinum compounds. For cisplatin-DNA, the rate constants (6.8 × 10 -5 and 6.5 × 10 -6 s -1 ) are assigned to the formation of monofunctional adducts and to bifunctional, intrastrand cross-linking, respectively. In transplatin-DNA, there is a 3.4-fold decrease in the rate constant of the slow phase, compared with the cisplatin samples. This change is attributed to generation of interstrand, rather than intrastrand, adducts. This longer reaction time may result in increased competition in the cellular environment and account, at least in part, for the lower pharmacological efficacy of transplatin.

Effects of glycation on meloxicam binding to human serum albumin

NASA Astrophysics Data System (ADS)

Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

2011-05-01

The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048.

PubMed

Silence, K; D'Hoore, A; Engelborghs, Y; Peyrot, V; Briand, C

1992-11-17

The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

NASA Astrophysics Data System (ADS)

Shahabadi, Nahid; Falsafi, Monireh

The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

Theory of long binding events in single-molecule–controlled rotation experiments on F1-ATPase

PubMed Central

Volkán-Kacsó, Sándor; Marcus, Rudolph A.

2017-01-01

The theory of elastic group transfer for the binding and release rate constants for nucleotides in F1-ATPase as a function of the rotor angle is further extended in several respects. (i) A method is described for predicting the experimentally observed lifetime distribution of long binding events in the controlled rotation experiments by taking into account the hydrolysis and synthesis reactions occurring during these events. (ii) A method is also given for treating the long binding events in the experiments and obtaining the rate constants for the hydrolysis and synthesis reactions occurring during these events. (iii) The theory in the previous paper is given in a symmetric form, an extension that simplifies the application of the theory to experiments. It also includes a theory-based correction of the reported “on” and “off” rates by calculating the missed events. A near symmetry of the data about the angle of −40° and a “turnover” in the binding rate data vs. rotor angle for angles greater than ∼40° is also discussed. PMID:28652332

Probing the binding of flavonoids to catalase by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Zhu, Jingfeng; Zhang, Xia; Li, Daojin; Jin, Jing

2007-10-01

The binding of flavonoids (quercetin and myricetin) to catalase was investigated by fluorescence and circular dichroism (CD) techniques under physiological conditions. The binding parameters and binding mode between flavonoids and catalase were determined, and the results of synchronous fluorescence spectra and CD indicated a conformational change of catalase with addition of flavonoids. The effect of both Cu 2+ and vitamin C on the binding constant of flavonoid-catalase was also examined. The experiment data show that the difference of the structure characteristics of quercetin and myricetin has a significant effect on their binding affinity for catalase.

Characterization of the interaction between furosemide and bovine serum albumin

NASA Astrophysics Data System (ADS)

Zhou, Neng; Liang, Yi-Zeng; Wang, Ping

2008-01-01

The interaction of furosemide (FU), one kind of potent diuretic, with bovine serum albumin (BSA) has been investigated at physiological acidity (pH 7.40) by fluorescent technique. Displacement experiment with site markers and Synchronous fluorescence clearly reveal that there are non-specific binding sites of FU with BSA. This conclusion was supported by the binding studies in the presence of the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and in different ionic strength. The binding sites number n and binding constant K were measured. The thermodynamic parameters Δ H°, Δ G°, Δ S° at different temperatures were calculated. The effects of other four diuretics, some common metal ions and bioactive components from herbal medicine on the binding are also considered. The results show only bumetanide has strong effect on FU's binding. Moreover, several data processing methods presently used were evaluated with Data of the same set. Quite different results were obtained from these methods suggesting more attention should be paid to the data processing methods.

Biophysical study on the interaction of ceftriaxone sodium with bovine serum albumin using spectroscopic methods.

PubMed

Pan, Jiongwei; Ye, Zaiting; Cai, Xiaoping; Wang, Liangxing; Cao, Zhuo

2012-12-01

The interaction of ceftriaxone sodium (CS), a cephalosporin antibiotic, with the major transport protein, bovine serum albumin (BSA), was investigated using different spectroscopic techniques such as fluorescence, circular dichroism (CD), and UV-vis spectroscopy. Values of binding parameters for BSA-CS interaction in terms of binding constant and number of binding sides were found to be 9.00 × 10(3), 3.24 × 10(3), and 2.30 × 10(3) M(-1) at 281, 301, and 321 K, respectively. Thermodynamic analysis of the binding data obtained at different temperatures showed that the binding process was spontaneous and was primarily mediated by van der Waals force or hydrogen bonding. CS binding to BSA caused secondary structural alterations in the protein as revealed by CD results. The distance between CS and Trp of BSA was determined as 3.23 nm according to the Förster resonance energy transfer theory. © 2012 Wiley Periodicals, Inc.

Biochemical investigation of yttrium(III) complex containing 1,10-phenanthroline: DNA binding and antibacterial activity.

PubMed

Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Moodi, Asieh; Niroomand, Sona

2013-03-05

Characterization of the interaction between yttrium(III) complex containing 1,10-phenanthroline as ligand, [Y(phen)2Cl(OH2)3]Cl2⋅H2O, and DNA has been carried out by UV absorption, fluorescence spectra and viscosity measurements in order to investigate binding mode. The experimental results indicate that the yttrium(III) complex binds to DNA and absorption is decreasing in charge transfer band with the increase in amount of DNA. The binding constant (Kb) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°), were calculated according to relevant fluorescent data and Vant' Hoff equation. The results of interaction mechanism studies, suggested that groove binding plays a major role in the binding of the complex and DNA. The activity of yttrium(III) complex against some bacteria was tested and antimicrobial screening tests shown growth inhibitory activity in the presence of yttrium(III) complex. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation.

PubMed

Zgliczyński, J M; Stelmaszyńska, T; Olszowska, E; Krawczyk, A; Kwasnowska, E; Wróbel, J T

1983-01-01

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.

Bidentate urea derivatives of p-tert-butyldihomooxacalix[4]arene: neutral receptors for anion complexation.

PubMed

Marcos, Paula M; Teixeira, Filipa A; Segurado, Manuel A P; Ascenso, José R; Bernardino, Raul J; Michel, Sylvia; Hubscher-Bruder, Véronique

2014-01-17

Three new bidentate ureidodihomooxacalix[4]arene derivatives (phenyl 5a, n-propyl 5b, and tert-butyl 5c) were synthesized in four steps from the parent compound p-tert-butyldihomooxacalix[4]arene and obtained in the cone conformation, as shown by NMR studies. The binding ability of these neutral receptors toward spherical, linear, trigonal planar, and tetrahedrical anions was assessed by (1)H NMR and UV-vis titrations. The structures and complexation energies of some complexes were also studied by DFT methods. The data showed that the association constants are strongly dependent on the nature of the substituent (aryl/alkyl) at the urea moiety. In general, for all the receptors, the association constants decrease with decrease of anion basicity. Ph-urea 5a is the best anion receptor, showing the strongest complexation for F(-) (log K(assoc) = 3.10 in CDCl3) and also high binding affinity for the carboxylates AcO(-) and BzO(-). Similar results were obtained by UV-vis studies and were also corroborated by DFT calculations.

Photoinduced interaction studies on N-(2-methylthiophenyl)-2-hydroxy-1-naphthadiamine with TiO2 nanoparticles: a combined experimental and theoretical (DFT and spectroscopic) approach.

PubMed

Pushpam, S; Gayathri, S; Ramakrishnan, V

2014-12-10

Schiff base derivative synthesized by the reaction of 2-(methylthio) aniline and 2-hydroxy-1-naphthaldehyde exhibits keto-amine tautomerism in methanol solvent. The fluorescence quenching of N-(2-methyl thiophenyl)-2-hydroxy-1-naphthadiamine (NMTHN) by TiO2 nanoparticles in methanol has been studied. The excitation and emission peaks have been observed at 439 and 509nm respectively. The apparent association constant has been deduced from the absorption spectral changes of NMTHN-TiO2 nanoparticles using Bensi-Hildebrand equation. The number of binding sites and the binding constant have been calculated from the relevant fluorescence data. Quenching of fluorescence of NMTHN by TiO2 could be due to a dynamic mode. Density Functional Theory (DFT) calculations also have been performed to study the charge distribution of NMTHN-TiO2 both in ground and excited states. The HOMO-LUMO analysis of NMTHN-TiO2 in the ground state has been made. Copyright © 2014 Elsevier B.V. All rights reserved.

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

PubMed Central

Young, Anne B.; Snyder, Solomon H.

1973-01-01

[3H]Strychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 μM, respectively. PMID:4200724

Exploring 3D structural influences of aliphatic and aromatic chemicals on α-cyclodextrin binding.

PubMed

Linden, Lukas; Goss, Kai-Uwe; Endo, Satoshi

2016-04-15

Binding of solutes to macromolecules is often influenced by steric effects caused by the 3D structures of both binding partners. In this study, the 1:1 α-cyclodextrin (αCD) binding constants (Ka1) for 70 organic chemicals were determined to explore the solute-structural effects on the αCD binding. Ka1 was measured using a three-part partitioning system with either a headspace or a passive sampler serving as the reference phase. The Ka1 values ranged from 1.08 to 4.97 log units. The results show that longer linear aliphatic chemicals form more stable complexes than shorter ones, and that the position of the functional group has a strong influence on Ka1, even stronger than the type of the functional group. Comparison of linear and variously branched aliphatic chemicals indicates that having a sterically unhindered alkyl chain is favorable for binding. These results suggest that only one alkyl chain can enter the binding cavity. Relatively small aromatic chemicals such as 1,3-dichlorobenzene bind to αCD well, while larger ones like tetrachlorobenzene and 3-ring aromatic chemicals show only a weak interaction with αCD, which can be explained by cavity exclusion. The findings of this study help interpret cyclodextrin binding data and facilitate the understanding of binding processes to macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

'Decoy peptide' region (RIFLKRMPSI) of prorenin prosegment plays a crucial role in prorenin binding to the (pro)renin receptor.

PubMed

Nabi, A H M Nurun; Biswas, Kazal Boron; Nakagawa, Tsutomu; Ichihara, Atsuhiro; Inagami, Tadashi; Suzuki, Fumiaki

2009-07-01

This study investigated a role of decoy peptide region (R10PIFLKRMPSI19P) in prorenin prosegment for prorenin binding to the (pro)renin receptor using the surface plasmon resonance technique. Three kinds of anti-receptor antibodies labeled as anti-107/121, anti-221/235 and anti-His tag antibody were prepared. The respective antigens D107SVANSIHSLFSEET121 (close to the N-terminal side of receptor), E221IGKRYGEDSEQFRD235 (N-terminal side of the transmembrane part of receptor) and 10xHis sequence (C-terminus) were designed based on the sequence of the receptor. These antibodies were immobilized on the CM5 sensor chip by amine coupling and allowed to bind to the receptor. Human prorenin, renin and the decoy bound to the receptor associated with antibodies. Their association (ka) and dissociation (kd) rate constants were measured and the dissociation constants (KD) were determined using Langmuir 1:1 kinetic binding model. The KD for interaction of prorenin and receptor associated to anti-107/121, anti-221/235 and anti-His tag antibodies were 2.9, 1.2 and 7.8 nM, respectively and for renin they were 9.3, 4.4 and 7.1 nM. The decoy bound to the respective immobilized receptor-antibody complexes at KD's of 6.2, 3.5 and 15.2 nM. Prorenin, renin and decoy had lower KD at the nanomolar ranges compared to those of L1PPTD4P in the prorenin prosegment and A248KKRLFDYVV257 in the C-domain of mature renin. The decoy reduced the binding of not only prorenin but also renin to (P)RR. These data are direct evidence that prorenin, renin and the peptides bind to (P)RR and the decoy reduces prorenin binding, supporting our hypothesis that decoy peptide region has a crucial role in prorenin binding.

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

PubMed

Romano, Edwin F; Quirino, Joselito P; Holdsworth, John L; So, Regina C; Holdsworth, Clovia I

2017-05-01

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K d 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins

NASA Astrophysics Data System (ADS)

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A.; Jiménez, M. Consuelo

2018-06-01

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α1-acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222.

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA

NASA Astrophysics Data System (ADS)

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-01

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (Kb) between TMG and DNA was 2.27 × 104 M- 1, that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH < 0 and ΔS < 0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking.

Studies on the interaction of a synthetic nitro-flavone derivative with DNA: A multi-spectroscopic and molecular docking approach.

PubMed

Mitra, A; Saikh, F; Das, J; Ghosh, S; Ghosh, R

2018-05-22

Interaction of a ligand with DNA is often the basis of drug action of many molecules. Flavones are important in this regard as their structural features confer them the ability to bind to DNA. 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO) is an important biologically active synthetic flavone derivative. We are therefore interested in studying its interaction with DNA. Absorption spectroscopy studies included standard and reverse titration, effect of ionic strength on titration, determination of stoichiometry of binding and thermal denaturation. Spectrofluorimetry techniques included fluorimetric titration, quenching studies and fluorescence displacement assay. Assessment of relative viscosity and estimation of thermodynamic parameters from CD spectral studies were also undertaken. Furthermore, molecular docking analyses were also done with different short DNA sequences. The fluorescent flavone 4NCO reversibly interacted with DNA through partial intercalation as well as minor-groove binding. The binding constant and the number of binding sites were of the order 10 4  M -1 and 1 respectively. The binding stoichiometry with DNA was found to be 1:1. The nature of the interaction of 4NCO with DNA was hydrophobic in nature and the process of binding was spontaneous, endothermic and entropy-driven. The flavone also showed a preference for binding to GC rich sequences. The study presents a profile for structural and thermodynamic parameters, for the binding of 4NCO with DNA. DNA is an important target for ligands that are effective against cell proliferative disorders. In this regard, the molecule 4NCO is important since it can exert its biological activity through its DNA binding ability and can be a potential drug candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanism of Na+ binding to thrombin resolved by ultra-rapid kinetics

PubMed Central

Gianni, Stefano; Ivarsson, Ylva; Bah, Alaji; Bush-Pelc, Leslie A.; Di Cera, Enrico

2007-01-01

The interaction of Na+ and K+ with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 μs we have been able to resolve the kinetic rate constants and entire mechanism of Na+ binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood. PMID:17935858

Studies on interaction of norbixin with DNA: Multispectroscopic and in silico analysis

NASA Astrophysics Data System (ADS)

Anantharaman, Amrita; Priya, Rajendra Rao; Hemachandran, Hridya; Sivaramakrishna, Akella; Babu, Subramanian; Siva, Ramamoorthy

2015-06-01

The interaction of food colorant norbixin with calf thymus DNA (CTDNA) was investigated through UV-Visible spectroscopy, Fourier Transform Infrared (FTIR), Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), DNA melting studies, electrophoretic analysis, histological staining technique and molecular docking studies. The results indicated that norbixin interacted with CTDNA by partial intercalation mode. The binding constant (K) of norbixin with CTDNA was calculated to be 5.08 × 105 Mol-1 L. FTIR and CD studies were coupled with 1H NMR spectra revealed that norbixin intercalates partially and binds to the groove's, phosphate group, deoxyribose sugar of DNA and also induces conformational transition of B-form to A-form DNA. Agarose gel electrophoretic and histological staining technique results further prove that, norbixin specifically binds to the DNA in the cell. Moreover, molecular docking studies on the specific binding of norbixin with CTDNA have exhibited lowest conformation energy score of -3.2. Therefore, this food colorant has the ability to interact with DNA and it could emerge as a promising class of natural DNA targeted therapeutic.

Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

PubMed

Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2017-01-20

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol -1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells.

PubMed

Serradeil-Le Gal, C; Herbert, J M; Delisee, C; Schaeffer, P; Raufaste, D; Garcia, C; Dol, F; Marty, E; Maffrand, J P; Le Fur, G

1995-01-01

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

The Role of Binding Site on the Mechanical Unfolding Mechanism of Ubiquitin

NASA Astrophysics Data System (ADS)

Cao, Penghui; Yoon, Gwonchan; Tao, Weiwei; Eom, Kilho; Park, Harold S.

2015-03-01

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between β5 and β3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution.

Sorption of Cr(III) and Cr(VI) to High and Low Pressure Synthetic Nano-Magnetite (Fe3O4)Particles

PubMed Central

Parsons, Jason G.; Hernandez, Jeffrey; Gonzalez, Christina M.; Gardea-Torresdey, J. L.

2014-01-01

The binding of Cr(III) and Cr(VI) to synthetic nano-magnetie particles synthesized under open vessel conditions and a microwave assisted hydrothermal synthesis techniques was investigated. Batch studies showed that the binding of both the Cr(III) and Cr(VI) bound to the nano-materials in a pH dependent manner. The Cr(III) maximized at binding at pH 4 and 100% binding. Similarly, the Cr(VI) ions showed a maximum binding of 100% at pH 4. The data from the time dependency studies showed for the most part the majority of the binding occurred within the first 5 minutes of contact with the nanomaterial and remained constant thereafter. In addition, the effects of the possible interferences were investigated which showed some effects on the binding of both Cr(III) and Cr(VI). However, the interferences never completely eliminated the chromium binding. Isotherm studies conducted at room temperature showed the microwave synthesized nanomaterials had a binding capacity of 1208 ± 43.9 mg/g and 555 ± 10.5 mg/g for Cr(VI) and Cr(III), respectively. However, the microwave assisted synthesized nanomaterials had capacities of 1705 ± 14.5 and 555± 10.5 mg/g for Cr(VI) and Cr(III), respectively. XANES studies showed the Cr(VI) was reduced to Cr(III), and the Cr(III) remained as Cr(III). In addition, the XANES studies indicated that the chromium remained coordinated in an octahedral arrangement of oxygen atoms. PMID:25097452

Equilibrium muscle cross-bridge behavior. Theoretical considerations.

PubMed Central

Schoenberg, M

1985-01-01

We have developed a model for the equilibrium attachment and detachment of myosin cross-bridges to actin that takes into account the possibility that a given cross-bridge can bind to one of a number of actin monomers, as seems likely, rather than to a site on only a single actin monomer, as is often assumed. The behavior of this multiple site model in response to constant velocity, as well as instantaneous stretches, was studied and the influence of system parameters on the force response explored. It was found that in the multiple site model the detachment rate constant has considerably greater influence on the mechanical response than the attachment rate constant. It is shown that one can obtain information about the detachment rate constants either by examining the relationship between the apparent stiffness and duration of stretch for constant velocity stretches or by examining the force-decay rate constants following an instantaneous stretch. The main effect of the attachment rate constant is to scale the mechanical response by influencing the number of attached cross-bridges. The significance of the modeling for the interpretation of experimental results is discussed. PMID:4041539

A detailed spectroscopic study on the interaction of Rhodamine 6G with human hemoglobin.

PubMed

Mandal, Paulami; Bardhan, Munmun; Ganguly, Tapan

2010-05-03

UV-vis, time-resolved fluorescence and circular dichroism spectroscopic investigations have been made to reveal the nature of the interactions between xanthene dye Rhodamine 6G and the well known protein hemoglobin. From the analysis of the steady-state and time-resolved fluorescence quenching of Rhodamine 6G in aqueous solutions in presence of hemoglobin, it is revealed that the quenching is static in nature. The primary binding pattern between Rhodamine and hemoglobin has been interpreted as combined effect of hydrophobic association and electrostatic interaction. The binding constants, number of binding sites and thermodynamic parameters at various pH of the environment have been computed. The binding average distance between the energy donor Rhodamine and acceptor hemoglobin has been determined from the Forster's theory. Copyright 2010 Elsevier B.V. All rights reserved.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

2005-11-01

The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

Tumor necrosis factor interaction with gold nanoparticles

NASA Astrophysics Data System (ADS)

Tsai, De-Hao; Elzey, Sherrie; Delrio, Frank W.; Keene, Athena M.; Tyner, Katherine M.; Clogston, Jeffrey D.; Maccuspie, Robert I.; Guha, Suvajyoti; Zachariah, Michael R.; Hackley, Vincent A.

2012-05-01

We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 +/- 0.02) nm-2 with a binding constant of 3 × 106 (mol L-1)-1. Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity.We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 +/- 0.02) nm-2 with a binding constant of 3 × 106 (mol L-1)-1. Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity. Electronic supplementary information (ESI) available: Experimental procedures, instrumentation, materials and calculations. See DOI: 10.1039/c2nr30415e

Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

PubMed Central

James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

2015-01-01

Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

PubMed

A, Ajith Kumar; Nadimpalli, Siva Kumar

2018-07-01

Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

Studies on the contributions of steric and polarity effects to the H2S-binding properties of Vitreoscilla hemoglobin

NASA Astrophysics Data System (ADS)

Wang, Dandan; Wang, Hui; Li, Haichao; Liu, Li; Li, Zhengqiang

2017-01-01

We have reported recently that Vitreoscilla hemoglobin (VHb) is a potential H2S receptor and storage molecule in bacterial metabolism. In this study, molecular cloning and site-directed mutagenesis were employed to investigate the structural basis for H2S binding. Association and dissociation rate constants (kon and koff) were determined using stopped-flow rapid-scanning spectrophotometry and compared with those for wild type VHb. Several unanticipated factors were found to govern H2S binding properties, due to the distinct structure of VHb. The results presented in this paper show that: i) bulkier residues at positions E7 and E11 decrease H2S binding accessibility, while the residue located at position B10 blocks bound H2S from escaping. ii) hydroxyl sidechains within the distal heme pocket reduce H2S reactivity to VHb; iii) Pro(E8) is involved in moving the E7-E10 loop region to trigger opening of the distal heme pocket to facilitate H2S binding.

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study.

PubMed

Meers, P; Feigenson, G W

1988-03-03

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.

Total-energy Assisted Tight-binding Method Based on Local Density Approximation of Density Functional Theory

NASA Astrophysics Data System (ADS)

Fujiwara, Takeo; Nishino, Shinya; Yamamoto, Susumu; Suzuki, Takashi; Ikeda, Minoru; Ohtani, Yasuaki

2018-06-01

A novel tight-binding method is developed, based on the extended Hückel approximation and charge self-consistency, with referring the band structure and the total energy of the local density approximation of the density functional theory. The parameters are so adjusted by computer that the result reproduces the band structure and the total energy, and the algorithm for determining parameters is established. The set of determined parameters is applicable to a variety of crystalline compounds and change of lattice constants, and, in other words, it is transferable. Examples are demonstrated for Si crystals of several crystalline structures varying lattice constants. Since the set of parameters is transferable, the present tight-binding method may be applicable also to molecular dynamics simulations of large-scale systems and long-time dynamical processes.

Ethidium and proflavine binding to a 2',5'-linked RNA duplex.

PubMed

Horowitz, Eric D; Hud, Nicholas V

2006-12-06

Despite over 40 years of physical investigations, fundamental questions persist regarding the energetics of RNA and DNA intercalation. The dramatic unwinding of a nucleic acid duplex upon intercalation immediately suggests that the nucleic acid backbone should play a significant role in dictating the free energy of intercalation. However, the contribution of the backbone to intercalation free energy is difficult to appreciate given the intertwined energetics associated with intercalation (e.g., pi-pi stacking and solvent effects). Fluorescence titrations were used to determine the association constants of two known intercalators, proflavine and ethidium, for duplex 2',5'-linked RNA. Proflavine was found to bind 2',5' RNA with an association constant 25-fold greater than that measured for standard, 3',5'-linked RNA. In contrast, ethidium binds 2',5' RNA less favorably than standard RNA.

Binding of Radioactive Benzylpenicillin to Sporulating Bacillus Cultures: Chemistry and Fluctuations in Specific Binding Capacity

PubMed Central

Lawrence, Paul J.; Rogolsky, Marvin; Hanh, Vo Thi

1971-01-01

The chemistry of the binding of 14C-benzylpenicillin to sporulating cultures of Bacillus megaterium and B. subtilis is similar to that in a 4-hr vegetative culture of Staphylococcus aureus. Unlabeled penicillins prevent the binding of 14C-benzylpenicillin, but benzylpenicilloic acid and benzylpenilloic acid do not. Bound antibiotic can be removed from cells with neutral hydroxylamine at 25 C. Sporulating cultures display two intervals of enhanced binding, whereas binding to stationaryphase S. aureus cells remains constant. The first period of increased binding activity occurs during formation of the spore septum or cell wall primordium development, and the second coincides with cortex biosynthesis. PMID:4942758

Fluorescence Spectroscopic Studies on the Complexation of Antidiabetic Drugs with Glycosylated Serum Albumin

NASA Astrophysics Data System (ADS)

Seedher, N.; Kanojia, M.

2013-11-01

Glycosylation decreases the association constant values and hence the binding affinity of human serum albumin (HSA) for the antidiabetic drugs under study. The percentage of HAS-bound drug at physiological temperature was only about 21-38 % as compared to 46-74 % for non-glycosylated HSA. Thus the percentage of free drug available for an antihyperglycemic effect was about double (62-79 %) compared to the values for non-glycosylated HSA. Much higher free drug concentrations available for pharmacological effect can lead to the risk of hypoglycemia. Hydrophobic interactions were predominantly involved in the binding. In the binding of gliclazide, hydrogen bonding and electrostatic interactions were involved. Site specificity for glycosylated HSA was the same as that for non-glycosylated HSA; gliclazide and repaglinide bind only at site II whereas glimepiride and glipizide bind at both sites I and II. Glycosylation, however, caused conformational changes in albumin, and the binding region within site II was different for glycosylated and non-glycosylated albumin. Stern-Volmer analysis also indicated the conformational changes in albumin as a result of glycosylation and showed that the dynamic quenching mechanism was valid for fluorescence of both glycosylated and non-glycosylated HSA.

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

2016-01-01

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

The effect of paracetamol on 5 fluorouracil and bovine serum albumin interaction: A biophysical study

NASA Astrophysics Data System (ADS)

Dahiya, Vandana; Pal, Samanwita

2018-05-01

Serum Albumin is a major carrier protein and its binding with drugs is important to examine the change in pharmacokinetic properties due to interaction amongst drugs. In the present study we have attempted to understand the relevant drug-drug interaction (DDI) between two common drugs viz, paracetamol, an anti-inflammatory and fluorouracil, an anti-cancer drug. In-vitro spectroscopic methods viz., fluorescence quenching and UV-vis absorption have been employed for the drug-bovine serum albumin (BSA) complexes studies. The binding parameters and quenching constants have been determined for BSA-Paracetamol and BSA-5Fluorouracil complex according to literature models. It is also predicted from the quenching studies that BSA-5Fluorouracil is a stronger complex than BSA-Paracetamol. On the other hand paracetamol can alter binding affinity of 5Fluorouracil towards BSA. Hence it becomes clear that although the drugs could be administered simultaneously but they influence each other's binding with protein in a concentration dependent fashion. Further these results also indicate that availability of free 5Fluorouracil in blood may increase in presence of paracetamol.

Magnetic properties and core electron binding energies of liquid water

NASA Astrophysics Data System (ADS)

Galamba, N.; Cabral, Benedito J. C.

2018-01-01

The magnetic properties and the core and inner valence electron binding energies of liquid water are investigated. The adopted methodology relies on the combination of molecular dynamics and electronic structure calculations. Born-Oppenheimer molecular dynamics with the Becke and Lee-Yang-Parr functionals for exchange and correlation, respectively, and includes an empirical correction (BLYP-D3) functional and classical molecular dynamics with the TIP4P/2005-F model were carried out. The Keal-Tozer functional was applied for predicting magnetic shielding and spin-spin coupling constants. Core and inner valence electron binding energies in liquid water were calculated with symmetry adapted cluster-configuration interaction. The relationship between the magnetic shielding constant σ(17O), the role played by the oxygen atom as a proton acceptor and donor, and the tetrahedral organisation of liquid water are investigated. The results indicate that the deshielding of the oxygen atom in water is very dependent on the order parameter (q) describing the tetrahedral organisation of the hydrogen bond network. The strong sensitivity of magnetic properties on changes of the electronic density in the nuclei environment is illustrated by a correlation between σ(17O) and the energy gap between the 1a1[O1s] (core) and the 2a1 (inner valence) orbitals of water. Although several studies discussed the eventual connection between magnetic properties and core electron binding energies, such a correlation could not be clearly established. Here, we demonstrate that for liquid water this correlation exists although involving the gap between electron binding energies of core and inner valence orbitals.

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc.

PubMed

Anderson, Charles T; Radford, Robert J; Zastrow, Melissa L; Zhang, Daniel Y; Apfel, Ulf-Peter; Lippard, Stephen J; Tzounopoulos, Thanos

2015-05-19

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling.

Combination of human acetylcholinesterase and serum albumin sensing surfaces as highly informative analytical tool for inhibitor screening.

PubMed

Fabini, Edoardo; Tramarin, Anna; Bartolini, Manuela

2018-06-05

In the continuous research for potential drug lead candidates, the availability of highly informative screening methodologies may constitute a decisive element in the selection of best-in-class compounds. In the present study, a surface plasmon resonance (SPR)-based assay was developed and employed to investigate interactions between human recombinant AChE (hAChE) and four known ligands: galantamine, tacrine, donepezil and edrophonium. To this aim, a sensor chip was functionalized with hAChE using mild immobilization conditions to best preserve enzyme integrity. Binding affinities and, for the first time, kinetic rate constants for all drug-hAChE complexes formation/disruption were determined. Inhibitors were classified in two groups: slow-reversible and fast-reversible binders according to respective target residence time. Combining data obtained on drug-target residence time with data obtained on serum albumin binding levels, a good correlation with potency, plasma protein binding in vivo, and administration regimen was found. The outcomes of this work demonstrated that the developed SPR-based assay is suitable for the screening, the binding affinity ranking and the kinetic evaluation of hAChE inhibitors. The method proposed ensures a simpler and cost-effective assay to quantify kinetic rate constants for inhibitor-hAChE interaction as compared with other proposed and published methods. Eventually, the determination of residence time in combination with preliminary ADME studies might constitute a better tool to predict in vivo behaviour, a key information for the research of new potential drug candidates. Copyright © 2018 Elsevier B.V. All rights reserved.

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc

PubMed Central

Anderson, Charles T.; Radford, Robert J.; Zastrow, Melissa L.; Zhang, Daniel Y.; Apfel, Ulf-Peter; Lippard, Stephen J.; Tzounopoulos, Thanos

2015-01-01

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling. PMID:25947151

Cerium Binding Activity of Pectins Isolated from the Seagrasses Zostera marina and Phyllospadix iwatensis

PubMed Central

Khotimchenko, Yuri; Khozhaenko, Elena; Kovalev, Valeri; Khotimchenko, Maxim

2012-01-01

Cerium binding activity of three different water soluble pectin compounds of different origin was studied in a batch sorption system. The Langmuir, Freundlich and BET sorption models were adopted to describe the binding reactions between metal ions and pectin molecules. The Langmuir model provided the best fit. Within the pH range from 4.0 to 6.0, the largest amount of the cerium ions was bound by pectin isolated from the seagrass Phylospadix iwatensis in comparison to pectin extracted from the seagrass Zostera marina and pectin obtained from citrus peel (commercial grade). The Langmuir constants were also highest for the pectin samples isolated from the seagrass P. iwatensis. The results obtained from this study suggest that pectin is a prospective source for the development of radioisotope-removing pharmaceuticals. PMID:22690146

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Organic additives stabilize RNA aptamer binding of malachite green.

PubMed

Zhou, Yubin; Chi, Hong; Wu, Yuanyuan; Marks, Robert S; Steele, Terry W J

2016-11-01

Aptamer-ligand binding has been utilized for biological applications due to its specific binding and synthetic nature. However, the applications will be limited if the binding or the ligand is unstable. Malachite green aptamer (MGA) and its labile ligand malachite green (MG) were found to have increasing apparent dissociation constants (Kd) as determined through the first order rate loss of emission intensity of the MGA-MG fluorescent complex. The fluorescent intensity loss was hypothesized to be from the hydrolysis of MG into malachite green carbinol base (MGOH). Random screening organic additives were found to reduce or retain the fluorescence emission and the calculated apparent Kd of MGA-MG binding. The protective effect became more apparent as the percentage of organic additives increased up to 10% v/v. The mechanism behind the organic additive protective effects was primarily from a ~5X increase in first order rate kinetics of MGOH→MG (kMGOH→MG), which significantly changed the equilibrium constant (Keq), favoring the generation of MG, versus MGOH without organic additives. A simple way has been developed to stabilize the apparent Kd of MGA-MG binding over 24h, which may be beneficial in stabilizing other triphenylmethane or carbocation ligand-aptamer interactions that are susceptible to SN1 hydrolysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding constants of phenylalanine for the four mononucleotides

NASA Technical Reports Server (NTRS)

Khaled, M. A.; Mullins, D. W., Jr.; Lacey, J. C., Jr.

1984-01-01

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: to which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.

PubMed

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

PubMed Central

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53. PMID:22162658

Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions.

PubMed

Lombardi, Dario; Dittrich, Petra S

2011-01-01

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug-protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps.

PubMed

Bradley, A J; Stoddart, D M

1992-01-01

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

Human blood-brain barrier insulin-like growth factor receptor

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

Duffy, K.R.; Pardridge, W.M.; Rosenfeld, R.G.

1988-02-01

Insulin-like growth factor (IGF)-1 and IGF-2, may be important regulatory molecules in the CNS. Possible origins of IGFs in brain include either de novo synthesis or transport of circulating IGFs from blood into brain via receptor mediated transcytosis mechanisms at the brain capillary endothelial wall, ie, the blood-brain barrier (BBB). In the present studies, isolated human brain capillaries are used as an in vitro model system of the human BBB and the characteristics of IGF-1 or IGF-2 binding to this preparation were assessed. The total binding of IGF-2 at 37 degrees C exceeded 130% per mg protein and was threefoldmore » greater than the total binding for IGF-1. However, at 37 degrees C nonsaturable binding equaled total binding, suggesting that endocytosis is rate limiting at physiologic temperatures. Binding studies performed at 4 degrees C slowed endocytosis to a greater extent than membrane binding, and specific binding of either IGF-1 or IGF-2 was detectable. Scatchard plots for either peptide were linear and the molar dissociation constant of IGF-1 and IGF-2 binding was 2.1 +/- 0.4 and 1.1 +/- 0.1 nmol/L, respectively. Superphysiologic concentrations of porcine insulin inhibited the binding of both IGF-1 (ED50 = 2 micrograms/mL) and IGF-2 (ED50 = 0.5 microgram/mL). Affinity cross linking of /sup 125/I-IGF-1, /sup 125/I-IGF-2, and /sup 125/I-insulin to isolated human brain capillaries was performed using disuccinimidylsuberate (DSS). These studies revealed a 141 kd binding site for both IGF-1 and IGF-2, and a 133 kd binding site for insulin.« less

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis

PubMed Central

Breuer, Christian; Kawamura, Ayako; Clark, Natalie M.; Morohashi, Kengo; Busch, Wolfgang; Benfey, Philip N.; Sozzani, Rosangela

2018-01-01

ABSTRACT How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis. Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors. PMID:29439132

Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

PubMed

Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

2014-07-18

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

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

Fuchs, G.; Mobassaleh, M.; Donohue-Rolfe, A.

This study examined the binding of purified /sup 125/I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4/sup 0/C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4/sup 0/C. Binding was inversely related to the temperature of incubation. A total of 80% ofmore » the labeled toxin binding at 4/sup 0/C dissociated from MVM when the temperature was raised to 37/sup 0/C, but reassociated when the temperature was again brought to 4/sup 0/C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.« less

Probing the Characterization of the Interaction of Aflatoxins B1 and G1 with Calf Thymus DNA In Vitro

PubMed Central

Ma, Liang; Wang, Jiaman; Zhang, Yuhao

2017-01-01

The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) bound to the ctDNA, forming complexes through hydrogen bonding. The binding constants of AFB1 and AFG1 with ctDNA reached up to 103 L·mol−1 and 104 L·mol−1, respectively, and AFG1 exhibited a higher binding propensity than that of AFB1. Furthermore, both AFB1 and AFG1 bound to the ctDNA through groove binding, as evidenced by the results of the spectroscopic, iodide quenching effect, viscosity, and ctDNA melting measurements. Changes in the circular dichroism signal manifested that both AFB1 and AFG1 induced an increase in the right-handed helicity, but only minimally influenced the base stacking of the DNA. A molecular docking study of the aflatoxin’s binding with the DNA revealed a groove binding mode, which was driven mainly by hydrogen bonding. This study of aflatoxin–ctDNA interaction may provide novel insights into the toxicological effect of the mycotoxins. PMID:28671585

Binding of a cyclic organoselenium compound with gold nanoparticles (GNP) and its effect on electron transfer properties.

PubMed

Kumar, Pavitra V; Singh, Beena G; Maiti, Nandita; Iwaoka, Michio; Priyadarsini, K Indira

2014-12-15

Binding of a cyclic organoselenium compound, DL-trans-3,4-dihydroxy-1-selenolane (DHSred) with gold nanoparticles (GNP) of different sizes was studied by absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), surface enhanced Raman spectroscopy (SERS) and zeta-potential (ζ) measurements. GNP of different size were synthesized by varying the reaction conditions and their size was determined by DLS and TEM techniques. The absorption spectral data showed red shift in the surface plasmon resonance (SPR) band indicating increase in the size of GNP on binding to DHSred. SERS studies confirmed that the binding of DHSred with GNP is through selenium center with planar orientation of DHSred on the GNP surface. The product of the number of binding sites (n) in GNP and the binding constant (K) was estimated for GNP of different particle size. The zeta potential (ζ) value of GNP decreased marginally in the presence of DHSred. Further, the binding of DHSred with GNP was found to enhance its reactivity with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS(·-)) and the reactivity increased with decrease in the GNP size. Such enhancement in the reducing ability may have a greater impact on the antioxidant activity of DHSred. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

PubMed

Zhang, Zijie; Liu, Juewen

2016-03-01

Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: advances toward treatment of hepatitis B virus infections.

PubMed

Shahabadi, Nahid; Falsafi, Monireh

2014-05-05

The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33±0.2×10(4) L mol(-1)and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH=34.4 kJ mol(-1); ΔS=184.32 J mol(-1) K(-1)). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol(-1). This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced spectrophotometric determination of Losartan potassium based on its physicochemical interaction with cationic surfactant

NASA Astrophysics Data System (ADS)

Abdel-Fattah, Laila; Abdel-Aziz, Lobna; Gaied, Mariam

2015-02-01

In this study, a simple and sensitive spectrophotometric method was developed for determination of Losartan potassium (LST K), an angiotensin-II receptor (type AT1) antagonist, in presence of cationic surfactant cetyltrimethylammonium bromide (CTAB). The physicochemical interaction of LST K with CTAB was investigated. The effect of cationic micelles on the spectroscopic and acid-base properties of LST K was studied at pH 7.4. The binding constant (Kb) and the partition coefficient (Kx) of LST K-CTAB were 1.62 × 105 M-1 and 1.38 × 105; respectively. The binding of LST K to CTAB micelles implied a shift in drug acidity constant (ΔpKa = 0.422). The developed method is linear over the range 0.5-28 μg mL-1. The accuracy was evaluated and was found to be 99.79 ± 0.509% and the relative standard deviation for intraday and interday precision was 0.821 and 0.963; respectively. The method was successfully applied to determine LST K in pharmaceutical formulations.

Enhanced spectrophotometric determination of Losartan potassium based on its physicochemical interaction with cationic surfactant.

PubMed

Abdel-Fattah, Laila; Abdel-Aziz, Lobna; Gaied, Mariam

2015-02-05

In this study, a simple and sensitive spectrophotometric method was developed for determination of Losartan potassium (LST K), an angiotensin-II receptor (type AT1) antagonist, in presence of cationic surfactant cetyltrimethylammonium bromide (CTAB). The physicochemical interaction of LST K with CTAB was investigated. The effect of cationic micelles on the spectroscopic and acid-base properties of LST K was studied at pH 7.4. The binding constant (Kb) and the partition coefficient (Kx) of LST K-CTAB were 1.62×10(5) M(-1) and 1.38×10(5); respectively. The binding of LST K to CTAB micelles implied a shift in drug acidity constant (ΔpKa=0.422). The developed method is linear over the range 0.5-28 μg mL(-1). The accuracy was evaluated and was found to be 99.79±0.509% and the relative standard deviation for intraday and interday precision was 0.821 and 0.963; respectively. The method was successfully applied to determine LST K in pharmaceutical formulations. Copyright © 2014 Elsevier B.V. All rights reserved.

Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

NASA Technical Reports Server (NTRS)

Kawamura, K.; Ferris, J. P.

1999-01-01

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

Alternate binding modes for a ubiquitin-SH3 domain interaction studied by NMR spectroscopy.

PubMed

Korzhnev, Dmitry M; Bezsonova, Irina; Lee, Soyoung; Chalikian, Tigran V; Kay, Lewis E

2009-02-20

Surfaces of many binding domains are plastic, enabling them to interact with multiple targets. An understanding of how they bind and recognize their partners is therefore predicated on characterizing such dynamic interfaces. Yet, these interfaces are difficult to study by standard biophysical techniques that often 'freeze' out conformations or that produce data averaged over an ensemble of conformers. In this study, we used NMR spectroscopy to study the interaction between the C-terminal SH3 domain of CIN85 and ubiquitin that involves the 'classical' binding sites of these proteins. Notably, chemical shift titration data of one target with another and relaxation dispersion data that report on millisecond time scale exchange processes are both well fit to a simple binding model in which free protein is in equilibrium with a single bound conformation. However, dissociation constants and chemical shift differences between free and bound states measured from both classes of experiment are in disagreement. It is shown that the data can be reconciled by considering three-state binding models involving two distinct bound conformations. By combining titration and dispersion data, kinetic and thermodynamic parameters of the three-state binding reaction are obtained along with chemical shifts for each state. A picture emerges in which one bound conformer has increased entropy and enthalpy relative to the second and chemical shifts similar to that of the free state, suggesting a less packed interface. This study provides an example of the interplay between entropy and enthalpy to fine-tune molecular interactions involving the same binding surfaces.

Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

NASA Astrophysics Data System (ADS)

Raman, N.; Sakthivel, A.; Pravin, N.

A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry†

PubMed Central

Prasannan, Charulata B.; Villar, Maria T.; Artigues, Antonio; Fenton, Aron W.

2013-01-01

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a non-allosteric analogue of the inhibitor. Alanine is used as the non-allosteric analogue since it binds competitively with phenylalanine, but elicits a negligible allosteric inhibition, i.e. a negligible reduction of the affinity of rM1-PYK for the substrate, phosphoenolpyruvate (PEP). This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e. those changes common upon the addition of alanine vs. phenylalanine) from changes associated with allosteric regulation (i.e. those elicited by the addition of phenylalanine binding, but not alanine binding). High quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique include residues previously identified by mutagenesis to have roles in the allosteric regulation by phenylalanine. PMID:23418858

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

PubMed Central

Tzafriri, A. Rami; Edelman, Elazer R.

2006-01-01

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered. PMID:17117924

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

PubMed

Mavelli, Fabio; Trotta, Massimo; Ciriaco, Fulvio; Agostiano, Angela; Giotta, Livia; Italiano, Francesca; Milano, Francesco

2014-07-01

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

Electrospray ionization mass spectrometric investigations of the complexation behavior of macrocyclic thiacrown ethers with bivalent transitional metals (Cu, Co, Ni and Zn).

PubMed

Tsybizova, Alexandra; Tarábek, Ján; Buchta, Michal; Holý, Petr; Schröder, Detlef

2012-10-15

Heavy metals are both a problem for the environment and an important resource for industry. Their selective extraction by means of organic ligands therefore is an attractive topic. The coordination of three thiacrown ethers to late 3d-metal ions was investigated by a combination of electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR). The mass spectrometric experiments were carried out in an ion trap mass spectrometer with an ESI source. Absolute binding constants were estimated by comparison with data for 18-crown-6/Na(+). EPR spectroscopy was used as a complementary method for investigating the Cu(I) /Cu(II) redox couple. The study found that thiacrown ethers preferentially bind traces of copper even at an excess of other metal ions (Co(II), Ni(II), and Zn(II)). The absolute association constants of the Cu(I) complexes were about 10(8) M(-1), and about two orders of magnitude lower for the other 3d-metal cations. The EPR spectra demonstrated that the reduction from Cu(II) to Cu(I) upon formation of the [(thiacrown)Cu](+) species takes place in solution. ESI-MS demonstrated that the three thiacrown ligands examined had high binding constants as well as good selectivities for copper(I) at low concentrations, and in the presence of other metal ions. By a combination of ESI-MS and EPR spectrometry it was shown that the reduction from Cu(II) to Cu(I) occurred in solution. Copyright © 2012 John Wiley & Sons, Ltd.

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

PubMed

Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

2015-10-01

The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

Competitive interactions of anti-carcinogens with serum albumin: a spectroscopic study of bendamustine and dexamethasone with the aid of chemometrics.

PubMed

Wang, Yong; Zhu, Ruirui; Ni, Yongnian; Kokot, Serge

2014-04-05

Interactions between the anti-carcinogens, bendamustine (BDM) and dexamethasone (DXM), with bovine serum albumin (BSA) were investigated with the use of fluorescence and UV-vis spectroscopies under pseudo-physiological conditions (Tris-HCl buffer, pH 7.4). The static mechanism was responsible for the fluorescence quenching during the interactions; the binding formation constant of the BSA-BDM complex and the binding number were 5.14×10(5)Lmol(-1) and 1.0, respectively. Spectroscopic studies for the formation of BDM-BSA complex were interpreted with the use of multivariate curve resolution - alternating least squares (MCR-ALS), which supported the complex formation. The BSA samples treated with site markers (warfarin - site I and ibuprofen - site II) were reacted separately with BDM and DXM; while both anti-carcinogens bound to site I, the binding constants suggested that DXM formed a more stable complex. Relative concentration profiles and the fluorescence spectra associated with BDM, DXM and BSA, were recovered simultaneously from the full fluorescence excitation-emission data with the use of the parallel factor analysis (PARAFAC) method. The results confirmed that on addition of DXM to the BDM-BSA complex, the BDM was replaced and the DXM-BSA complex formed; free BDM was released. This finding may have consequences for the transport of these drugs during any anti-cancer treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

Material Science Smart Coatings

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

Rubinstein, A. I.; Sabirianov, R. F.; Namavar, Fereydoon

2014-07-01

The contribution of electrostatic interactions to the free energy of binding between model protein and a ceramic implant surface in the aqueous solvent, considered in the framework of the nonlocal electrostatic model, is calculated as a function of the implant low-frequency dielectric constant. We show that the existence of a dynamically ordered (low-dielectric) interfacial solvent layer at the protein-solvent and ceramic-solvent interface markedly increases charging energy of the protein and ceramic implant, and consequently makes the electrostatic contribution to the protein-ceramic binding energy more favorable (attractive). Our analysis shows that the corresponding electrostatic energy between protein and oxide ceramics dependsmore » nonmonotonically on the dielectric constant of ceramic, ε C. Obtained results indicate that protein can attract electrostatically to the surface if ceramic material has a moderate ε C below or about 35 (in particularly ZrO 2 or Ta 2O 5). This is in contrast to classical (local) consideration of the solvent, which demonstrates an unfavorable electrostatic interaction of protein with typical metal oxide ceramic materials (ε C>10). Thus, a solid implant coated by combining oxide ceramic with a reduced dielectric constant can be beneficial to strengthen the electrostatic binding of the protein-implant complex.« less

Diffusion Monte Carlo method for evaluating Hamaker constants

NASA Astrophysics Data System (ADS)

Maezono, Ryo; Hongo, Kenta

We evaluated Hamaker's constants for Si6H12 (CHS) to investigate its wettability, which is industrially useful but no references available. The constant is fundamental for wettability, but not directly accessible by experiments. Ab initio estimations are therefore in demand, and surely give an impact for broader fields such as tribology where the wettability plays an important role. The evaluation of binding curves itself could be a big challenge if it is applied to a practical molecule such as CHS, because highly accurate descriptions of electron correlations in vdW bindings get tough for such larger sizes with anisotropy. We applied DMC to overcome this difficulty, showing a new direction for wettability issues. Since ab intio estimations rely on simple assumptions such as additivity (and hence we denote it as Aadd), it would include biases. Taking a benzene as a benchmark, we compared Aadd evaluated from several available binding curves with other reported AL (estimations based on Lifshitz theory). By the comparison, we get trends of biases in Aa dd due to non-additivity and anisotropy because AL is expected to capture these effects to some extent in macroscopic manner. The expected trends here surprisingly well explain the series of results for CHS.

Osmotically-induced tension and the binding of N-BAR protein to lipid vesicles.

PubMed

Hutchison, Jaime B; Karunanayake Mudiyanselage, Aruni P K K; Weis, Robert M; Dinsmore, Anthony D

2016-02-28

The binding affinity of a curvature-sensing protein domain (N-BAR) is measured as a function of applied osmotic stress while the membrane curvature is nearly constant. Varying the osmotic stress allows us to control membrane tension, which provides a probe of the mechanism of binding. We study the N-BAR domain of the Drosophila amphiphysin and monitor its binding on 50 nm-radius vesicles composed of 90 mol% DOPC and 10 mol% PIP. We find that the bound fraction of N-BAR is enhanced by a factor of approximately 6.5 when the tension increases from zero to 2.6 mN m(-1). This tension-induced response can be explained by the hydrophobic insertion mechanism. From the data we extract a hydrophobic domain area that is consistent with known structure. These results indicate that membrane stress and strain could play a major role in the previously reported curvature-affinity of N-BAR.

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC.

PubMed

Su, Xingye; Kong, Liang; Li, Xin; Chen, Xueguo; Guo, Ming; Zou, Hanfa

2005-05-27

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.

Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

NASA Astrophysics Data System (ADS)

Siman, L.; Carrasco, I. S. S.; da Silva, J. K. L.; de Oliveira, M. C.; Rocha, M. S.; Mesquita, O. N.

2012-12-01

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

PubMed

Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

2015-01-01

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Quantitation of Membrane-Ligand Interactions Using Backscattering Interferometry

PubMed Central

Baksh, Michael M.; Kussrow, Amanda K.; Mileni, Mauro; Finn, M.G.; Bornhop, Darryl J.

2011-01-01

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest. PMID:21399645

Spectroscopic studies on the interactions between 3,4-dihydropyrimidin-2(1H)-ones and bovine serum albumin.

PubMed

Yu, Xianyong; Liu, Ronghua; Ji, Danhong; Xie, Jian; Yang, Fengxian; Li, Xiaofang; Huang, Haowen; Yi, Pinggui

2010-09-15

The interactions between 3,4-dihydropyrimidin-2(1H)-ones (DHPM) and bovine serum albumin (BSA) were investigated by fluorescence and ultraviolet spectroscopy under imitated physiological conditions. The experimental results showed that all DHPM could form complexes with BSA. Static quenching and non-radiation energy transfer are the main reasons leading to the fluorescence quenching. The binding constants (K(A)) and the number of binding sites (n) were calculated. According to Förster theory of non-radiation energy transfer, the binding distances (r) between BSA and DHPM are less than 7 nm. The relationship between different aryl groups in pyrimidine ring and the binding ability of DHPM with BSA is preliminarily discussed. Moreover, the synchronous fluorescence spectra indicated that the conformation of BSA has not been changed. Copyright 2010 Elsevier B.V. All rights reserved.

Self-assembling choline mimicks with enhanced binding affinities to C-LytA protein

PubMed Central

Shi, Yang; Zhou, Hao; Zhang, Xiaoli; Wang, Jingyu; Long, Jiafu; Yang, Zhimou; Ding, Dan

2014-01-01

Streptococcus pneumoniae (pneumococcus) causes multiple illnesses in humans. Exploration of effective inhibitors with multivalent attachment sites for choline-binding modules is of great importance to reduce the pneumococcal virulence. In this work, we successfully developed two self-assembling choline mimicks, Ada-GFFYKKK' and Nap-GFFYKKK', which have the abilities to self-assemble into nanoparticles and nanofibers, respectively, yielding multivalent architectures. Additionally, the best characterized choline-binding module, C-terminal moiety of the pneumococcal cell-wall amidase LytA (C-LytA) was also produced with high purity. The self-assembling Ada-GFFYKKK' and Nap-GFFYKKK' show strong interactions with C-LytA, which possess much higher association constant values to the choline-binding modules as compared to the individual peptide Fmoc-K'. This study thus provides a self-assembly approach to yield inhibitors that are very promising for reducing the pneumococcal virulence. PMID:25315737

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

PubMed Central

Qian, Yufeng; Johnson, Kenneth A.

2017-01-01

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m−1 s−1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. PMID:28615444

Facilitated release of substrate protein from prefoldin by chaperonin.

PubMed

Zako, Tamotsu; Iizuka, Ryo; Okochi, Mina; Nomura, Tomoko; Ueno, Taro; Tadakuma, Hisashi; Yohda, Masafumi; Funatsu, Takashi

2005-07-04

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin.

Prototype Development and Dynamic Characterization of Deployable CubeSat Booms

DTIC Science & Technology

2010-03-01

constant force of gravity and the constant force of photons impinging on the reflective Mylar surface of the craft. This could, in effect, provide a much...reflected photons of light for spacecraft propulsion. As acceleration is inversely proportional to the mass for a constant thrust, this method of...of the satellite. Additionally, with so much boom essentially stuffed within a small cavity, binding and entanglement issues are a near certainty

Interaction of the recently approved anticancer drug nintedanib with human acute phase reactant α 1-acid glycoprotein

NASA Astrophysics Data System (ADS)

Abdelhameed, Ali Saber; Ajmal, Mohammad Rehan; Ponnusamy, Kalaiarasan; Subbarao, Naidu; Khan, Rizwan Hasan

2016-07-01

A comprehensive study of the interaction of the newly approved tyrosine kinase inhibitor, Nintedanib (NTB) and Alpha-1 Acid Glycoprotein (AAG) has been carried out by utilizing UV-Vis spectroscopy, fluorescence spectroscopy, circular dichroism, dynamic light scattering and molecular docking techniques. The obtained results showed enhancement of the UV-Vis peak of the protein upon binding to NTB with the fluorescence intensity of AAG is being quenched by NTB via the formation of ground state complex (i.e. Static quenching). Forster distance (Ro) obtained from fluorescence resonance energy transfer (FRET) is found to be 2.3 nm. The calculated binding parameters from the modified Stern-Volmer equation showed that NTB binds to AAG with a binding constant in the order of 103. Conformational alteration of the protein upon its binding to NTB was confirmed by the circular dichroism. Dynamic light scattering results showed that the binding interaction of NTB leads to the reduction in hydrodynamic radii of AAG. Dynamic molecular docking results showed that the NTB fits into the central binding cavity in AAG and hydrophobic interaction played the key role in the binding process also the docking studies were performed with methotrexate and clofarabine drugs to look into the common binding regions of these drugs on AAG molecule, it was found that five amino acid residues namely Phe 113, Arg 89, Tyr 126, Phe 48 and Glu 63 were common among the binding regions of three studied drugs this phenomenon of overlapping binding regions may influence the drug transport by the carrier molecule in turn affecting the metabolism of the drug and treatment outcome.

Investigation on potential enzyme toxicity of clenbuterol to trypsin

NASA Astrophysics Data System (ADS)

Chai, Jun; Xu, Qifei; Dai, Jinping; Liu, Rutao

2013-03-01

Clenbuterol (CLB) is a kind of β2-adrenergic agonists which was illegally used as feed additives nowadays. The toxic interaction of CLB with trypsin, an important digestive enzyme, was studied in vitro using multi-spectroscopic methods and molecular modeling methods. CLB was proved to bind with trypsin in S1 pocket, forming a complex driven by the dominant force of H-bond. The binding constant was calculated to be 1.79887 × 105 L mol-1 at 289 K and 0.32584 × 105 L mol-1 at 310 K, respectively. The skeleton of trypsin became loosened and unfolded with the amino residues microenvironment changed. The secondary and tertiary structure of trypsin also varied. Molecular modeling studies illustrated specific display of the binding information and explained most of the experiment phenomena. The binding site of CLB induced the fluorescence quenching as well as inhibition of enzyme activity of trypsin. The study confirmed that CLB had potential toxicity on both the structure and function of trypsin and the effects enhanced with the increasing concentration of CLB.

Spectrofluorimetric and molecular docking studies on the interaction of cyanidin-3-O-glucoside with whey protein, β-lactoglobulin.

PubMed

Cheng, Jing; Liu, Jian-Hua; Prasanna, Govindarajan; Jing, Pu

2017-12-01

The interaction of β-Lactoglobulin (β-Lg) with cyanidin-3-O-glucoside (C3G) was characterized using fluorescence, circular dichroism spectroscopy, and docking studies under physiological conditions. Fluorescence studies showed that β-Lg has a strong binding affinity for C3G via hydrophobic interaction with the binding constant, K a , of 3.14×10 4 M -1 at 298K. The secondary structure of β-Lg displayed an increase in the major structure of β-sheet upon binding with C3G, whereas a decrease in the minor structure of α-helix was also observed. In addition, evidenced by near UV-CD, the interaction also disrupted the environments of Trp residues. The molecular docking results illustrated that both hydrogen bonding and the hydrophobic interaction are involved as an acting force during the binding process. These results may contribute to a better understanding over the enhanced physicochemical proprieties of anthocyanins due to the complexation with milk proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Milk β-casein as a vehicle for delivery of bis(indolyl)methane: Spectroscopy and molecular docking studies

NASA Astrophysics Data System (ADS)

Dezhampanah, Hamid; Esmaili, Masoomeh; Khorshidi, Alireza

2017-05-01

The interaction of bis(indolyl)methane with bovine milk β-casein was investigated using spectroscopy and molecular docking studies at different temperatures (25-37 °C). The circular dichroism and Fourier transform infrared spectroscopic data demonstrated that β-casein interacts with BIM molecule mainly via both the hydrophobic and hydrophilic interactions with a minor change in the secondary structure of β-casein. The fluorescence quenching measurements revealed that the presence of a single binding site on β-casein for BIM with the binding constant value of ∼104 M-1. The negative values of entropy and enthalpy changes confirm the predominate role of hydrogen binding and van der Waals interactions in the binding process. Fӧrster energy transfer measurement suggested that the distance between bound BIM and Trp residue is higher than the respective critical distance. Hence, the static quenching is more likely responsible for the fluorescence quenching rather than the mechanism of non-radiative. Docking study showed that BIM molecule forms three hydrogen bonds and several van der Waals contacts with β-casein.

The relationship between metal toxicity and biotic ligand binding affinities in aquatic and soil organisms: a review.

PubMed

Ardestani, Masoud M; van Straalen, Nico M; van Gestel, Cornelis A M

2014-12-01

The biotic ligand model (BLM) is a theoretical, potentially mechanistic approach to assess metal bioavailability in soil and aquatic systems. In a BLM, toxicity is linked to the fraction of biotic ligand occupied, which in turn, depends on the various components of the solution, including activity of the metal. Bioavailability is a key factor in determining toxicity and uptake of metals in organisms. In this study, the present status of BLM development for soil and aquatic organisms is summarized. For all species and all metals, toxicity was correlated with the conditional biotic ligand binding constants. For almost all organisms, values for Ag, Cu, and Cd were higher than those for Zn and Ni. The constants derived for aquatic systems seem to be equally valid for soil organisms, but in the case of soils, bioavailability from the soil solution is greatly influenced by the presence of the soil solid phase. Copyright © 2014 Elsevier Ltd. All rights reserved.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

PubMed

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Binding of mitomycin C to blood proteins: A spectroscopic analysis and molecular docking

NASA Astrophysics Data System (ADS)

Jang, Jongchol; Liu, Hui; Chen, Wei; Zou, Guolin

2009-06-01

Mitomycin C (MMC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. The binding of MMC to two human blood proteins, human serum albumin (HSA) and human hemoglobin (HHb), have been investigated by fluorescence quenching, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking methods. The fluorescence data showed that binding of MMC to proteins caused strong fluorescence quenching of proteins through a static quenching way, and each protein had only one binding site for the drug. The binding constants of MMC to HSA and HHb at 298 K were 2.71 × 10 4 and 2.56 × 10 4 L mol -1, respectively. Thermodynamic analysis suggested that both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HSA or HHb. The CD spectroscopy indicated that the secondary structures of the two proteins were not changed in the presence of MMC. The study of molecular docking showed that MMC was located in the entrance of site I of HSA, and in the central cavity of HHb.

Imino proton exchange and base-pair kinetics in the AMP-RNA aptamer complex.

PubMed

Nonin, S; Jiang, F; Patel, D J

1997-05-02

We report on the dynamics of base-pair opening in the ATP-binding asymmetric internal loop and flanking base-pairs of the AMP-RNA aptamer complex by monitoring the exchange characteristics of the extremely well resolved imino protons in the NMR spectrum of the complex. The kinetics of imino proton exchange as a function of basic pH or added ammonia catalyst are used to measure the apparent base-pair dissociation constants and lifetimes of Watson-Crick and mismatched base-pairs, as well as the solvent accessibility of the unpaired imino protons in the complex. The exchange characteristics of the imino protons identify the existence of four additional hydrogen bonds stabilizing the conformation of the asymmetric ATP-binding internal loop that were not detected by NOEs and coupling constants alone, but are readily accommodated in the previously reported solution structure of the AMP-RNA aptamer complex published from our laboratory. The hydrogen exchange kinetics of the non-Watson-Crick pairs in the asymmetric internal loop of the AMP-RNA aptamer complex have been characterized and yield apparent dissociation constants (alphaKd) that range from 10(-2) to 10(-7). Surprisingly, three of these alphaKd values are amongst the lowest measured for all base-pairs in the AMP-RNA aptamer complex. Comparative studies of hydrogen exchange of the imino protons in the free RNA aptamer and the AMP-RNA aptamer complex establish that complexation stabilizes not only the bases within the ATP-binding asymmetric internal loop, but also the flanking stem base-pairs (two pairs on either side) of the binding site. We also outline some preliminary results related to the exchange properties of a sugar 2'-hydroxyl proton of a guanosine residue involved in a novel hydrogen bond that has been shown to contribute to the immobilization of the bound AMP by the RNA aptamer, and whose resonance is narrow and downfield shifted in the spectrum.

Effective binding of perhalogenated closo-borates to serum albumins revealed by spectroscopic and ITC studies

NASA Astrophysics Data System (ADS)

Kuperman, Marina V.; Losytskyy, Mykhaylo Yu.; Bykov, Alexander Yu.; Yarmoluk, Sergiy M.; Zhizhin, Konstantin Yu.; Kuznetsov, Nikolay T.; Varzatskii, Oleg A.; Gumienna-Kontecka, Elzbieta; Kovalska, Vladyslava B.

2017-08-01

The interactions of boron cluster compounds closo-borates with biomolecules are widely studied due to their efficiency as agents for boron neutron capture therapy of cancer. In present work the binding abilities of anionic halogen closo-borates [B10Hal10]2- (Hal = Cl, Br, I) and [B12Hal12]2- (Hal = Cl, I) towards bovine and human serum albumins were investigated by spectroscopic and isothermal titration calorimetry (ITC) methods. The protein fluorescence quenching method and ITC studies confirmed the complex formation. The degree of protein fluorescence quenching increased from chlorine to iodine boron derivatives that is attributed to external heavy atom effect. The ITC data point on the existence in the protein structure of two types of binding sites: with higher and lower affinity to closo-borates. Albumin-closo-borate complex binding ratio, n (4-5 anions per protein molecule) is higher than for the parent hydrogen closo-borates (2 anions per protein molecule). Binding constants estimated by fluorescent and ITC methods indicate higher affinity of halogen closo-borates to albumins (K in the range of 104-106 M-1) comparing to that of the hydrogen closo-borate (K about 103 M-1). Due to their high affinity and high binding ratio to albumins halogen closo-borates are proposed for further studies as agents for boron neutron capture therapy.

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins.

PubMed

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2018-06-15

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α 1 -acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222. Copyright © 2018 Elsevier B.V. All rights reserved.

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA.

PubMed

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-05

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (K b ) between TMG and DNA was 2.27×10 4 M -1 , that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH<0 and ΔS<0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors.

PubMed Central

Alexander, M D; Andrews, J A; Leslie, R G; Wood, N J

1978-01-01

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2. PMID:680795

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

NASA Astrophysics Data System (ADS)

Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

2014-06-01

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

Thermodynamic and Spectroscopic Studies of Trivalent f -element Complexation with Ethylenediamine- N,N '-di(acetylglycine)- N,N '-diacetic Acid

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

Heathman, Colt R.; Grimes, Travis S.; Zalupski, Peter R.

In this study, the coordination behavior and thermodynamic features of complexation of trivalent lanthanides and americium by ethylenediamine- N,N'-di(acetylglycine)- N,N'-diacetic acid (EDDAG-DA) (bisamide-substituted-EDTA) were investigated by potentiometric and spectroscopic techniques. Acid dissociation constants (K a) and complexation constants (β) of lanthanides (except Pm) were determined by potentiometric analysis. Absorption spectroscopy was used to determine stability constants for the binding of trivalent americium and neodymium by EDDAG-DA under similar conditions. The potentiometry revealed 5 discernible protonation constants and 3 distinct metal–ligand complexes (identified as ML –, MHL, and MH 2L +). Time-resolved fluorescence studies of Eu-(EDDAG-DA) solutions (at varying pH) identifiedmore » a constant inner-sphere hydration number of 3, suggesting that glycine functionalities contained in the amide pendant arms are not involved in metal complexation and are protonated under more acidic conditions. The thermodynamic studies identified that f-element coordination by EDDAG-DA is similar to that observed for ethylenediamine- N,N,N',N'-tetraacetic acid (EDTA). However, coordination via two amidic oxygens of EDDAG-DA lowers its trivalent f-element complex stability by roughly 3 orders of magnitude relative to EDTA.« less

Thermodynamic and Spectroscopic Studies of Trivalent f -element Complexation with Ethylenediamine- N,N '-di(acetylglycine)- N,N '-diacetic Acid

DOE PAGES

Heathman, Colt R.; Grimes, Travis S.; Zalupski, Peter R.

2016-03-21

In this study, the coordination behavior and thermodynamic features of complexation of trivalent lanthanides and americium by ethylenediamine- N,N'-di(acetylglycine)- N,N'-diacetic acid (EDDAG-DA) (bisamide-substituted-EDTA) were investigated by potentiometric and spectroscopic techniques. Acid dissociation constants (K a) and complexation constants (β) of lanthanides (except Pm) were determined by potentiometric analysis. Absorption spectroscopy was used to determine stability constants for the binding of trivalent americium and neodymium by EDDAG-DA under similar conditions. The potentiometry revealed 5 discernible protonation constants and 3 distinct metal–ligand complexes (identified as ML –, MHL, and MH 2L +). Time-resolved fluorescence studies of Eu-(EDDAG-DA) solutions (at varying pH) identifiedmore » a constant inner-sphere hydration number of 3, suggesting that glycine functionalities contained in the amide pendant arms are not involved in metal complexation and are protonated under more acidic conditions. The thermodynamic studies identified that f-element coordination by EDDAG-DA is similar to that observed for ethylenediamine- N,N,N',N'-tetraacetic acid (EDTA). However, coordination via two amidic oxygens of EDDAG-DA lowers its trivalent f-element complex stability by roughly 3 orders of magnitude relative to EDTA.« less

Quantifying Na(I)-insulin and K(I)-insulin non-covalent complexes by ESI-MS method and calculation of their equilibrium constants.

PubMed

Gülfen, Mustafa; Özdemir, Abdil; Lin, Jung-Lee; Chen, Chung-Hsuan

2017-10-01

In this study, the dissociation and formation equilibrium constants of Na(I)-insulin and K(I)-insulin complexes have been calculated after the quantifying them on ESI mass spectrometer. The ESI-MS spectra of the complexes were measured by using the solvents as 50% MeOH in water and 100% water. The effect of pH on the Na(I)-insulin and K(I)-insulin complex formation were examined. Serial binding of Na(I) and K(I) ions to the insulin molecule were observed in the ESI-MS measurements. The first formation equilibrium constants were calculated as K f1 : 5.48×10 3 1/M for Na(I)-insulin complex and K f1 : 4.87×10 3 1/M for K(I)-insulin in water. The binding capability of Na(I) ions to insulin molecule is higher than the capability of K(I) ions. In case of a comparison together with Ca(II)-insulin and Mg(II)-insulin, the formation equilibrium constants (K f1 ) are in order of Ca(II)-insulin>Mg(II)-insulin>Na(I)-insulin>K(I)-insulin in water. The results showed that Na(I) and K(I) ions are involved in the formation of the non-covalent complexes with insulin molecule, since high extracellular and intracellular concentrations of them in the body. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction of glucocorticoids and progesterone derivatives with human serum albumin.

PubMed

Abboud, Rola; Akil, Mohammad; Charcosset, Catherine; Greige-Gerges, Hélène

2017-10-01

Glucocorticoids (GCs) and progesterone derivatives (PGDs) are steroid hormones with well-known biological activities. Their interaction with human serum albumin (HSA) may control their distribution. Their binding to albumin is poorly studied in literature. This paper deals with the interaction of a series of GCs (cortisol, cortisone, prednisolone, prednisone, 6-methylprednisolone and 9-fluorocortisol acetate) and PGDs (progesterone, hydroxylated PGDs, methylated PGDs and dydrogesterone) with HSA solution (pH 7.4) at molar ratios steroid to HSA varying from 0 to 10. Similar titrations were conducted using Trp aqueous solution. Fluorescence titration method and Fourier transform infrared spectroscopy (FTIR) are used. PGDs (except dydrogesterone), cortisone and 9-fluorocortisol acetate affected weakly the fluorescence of Trp in buffer solution while they decreased in a dose-dependent manner that of HSA. Their binding constants to HSA were then calculated. Moreover, displacement experiment was performed using bilirubin as a site marker. The binding constant of bilirubin to albumin was determined in the absence and presence of a steroid at a molar ratio steroid to HSA of 1. The results indicate that the steroids bind to HSA at site I in a pocket different from that of bilirubin. Furthermore, the peak positions of amide I and amide II bands of HSA were shifted in the presence of progesterone, dydrogesterone and GCs. Also a variation was observed in amide I region indicating the formation of hydrogen bonding between albumin and steroids. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding selectivity of vitamin K3 based chemosensors towards nickel(II) and copper(II) metal ions

NASA Astrophysics Data System (ADS)

Patil, Amit; Lande, Dipali N.; Nalkar, Archana; Gejji, Shridhar P.; Chakrovorty, Debamitra; Gonnade, Rajesh; Moniz, Tânia; Rangel, Maria; Pereira, Eulália; Salunke-Gawali, Sunita

2017-09-01

The vitamin K3 derivatives 2-methyl-3-[(pyridin-2-ylmethyl)-amino]-1,4-naphthoquinone (M-1), 2-methyl-3-[(pyridin-2-ylethyl)-amino]-1,4-naphthoquinone (M-2), 2-methyl-3-((2-(thiophen-2-yl)methyl)amino)naphthalene-1,4-dione (M-3) and 2-methyl-3-((2-(thiophen-2-yl)ethyl)amino)naphthalene-1,4-dione (M-4) have been synthesized, characterized and studied for their chemosensor abilities towards transition metal ions. Crystal structures of M-1 to M-4 revealed a variety of Nsbnd H⋯O, Csbnd H⋯O, Csbnd H⋯π and π⋯π interactions. Minor variations in such interactions by chemical stimuli such as metal ions, results in change in color that can be visualized by naked eyes. It has been shown that electronic structure and 1H NMR, vibrational as well as electronic spectra from the density functional theory agree well with the experiments. The metal ion binding in ethanol, ethanol-water and in mild base triethylamine brings forth recognizing ability of M-1 toward Ni2+ whereas M-2 exhibits large sensing ability for Cu2+ ion. Interestingly M-1 display varying metal ion binding specificity in different solvents with the association constant in ethanol being 11,786 M-1 for Ni2+ compared to 9462 M-1 for the Cu2+. A reversal in preferential binding of M-2 with the respective association constants being 4190 M-1 and 6370 M-1 is discernible.

Characterization of inclusion complexes of organic ions with hydrophilic hosts by ion transfer voltammetry with solvent polymeric membranes.

PubMed

Olmos, José Manuel; Laborda, Eduardo; Ortuño, Joaquín Ángel; Molina, Ángela

2017-03-01

The quantitative characterization of inclusion complexes formed in aqueous phase between organic ions and hydrophilic hosts by ion-transfer voltammetry with solvent polymeric membrane ion sensors is studied, both in a theoretical and experimental way. Simple analytical solutions are presented for the determination of the binding constant of the complex from the variation with the host concentration of the electrochemical signal. These solutions are valid for any voltammetric technique and for solvent polymeric membrane ion sensors comprising one polarisable interface (1PI) and also, for the first time, two polarisable interfaces (2PIs). Suitable experimental conditions and data analysis procedures are discussed and applied to the study of the interactions of a common ionic liquid cation (1-octyl-3-metyl-imidazolium) and an ionisable drug (clomipramine) with two hydrophilic cyclodextrins: α-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. The experimental study is performed via square wave voltammetry with 2PIs and 1PI solvent polymeric membranes and in both cases the electrochemical experiments enable the detection of inclusion complexes and the determination of the corresponding binding constant. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence spectroscopic and molecular docking studies of the binding interaction between the new anaplastic lymphoma kinase inhibitor crizotinib and bovine serum albumin

NASA Astrophysics Data System (ADS)

Abdelhameed, Ali S.; Alanazi, Amer M.; Bakheit, Ahmed H.; Darwish, Hany W.; Ghabbour, Hazem A.; Darwish, Ibrahim A.

2017-01-01

Binding of the recently introduced anti-cancer drug, crizotinib (CRB) with the bovine serum albumin (BSA) was comprehensively studied with the aid of fluorescence and UV-Vis spectroscopic as well as molecular docking techniques. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the CRB and BSA with binding constants of 104 L mol- 1. BSA conformational changes were investigated using three dimensional (3D) and synchronous fluorescence measurements. Moreover, the results of site marker competitive experiments and molecular docking, it could be deduced that CRB was inserted into the subdomain IIA (site I) of BSA yielding a more stabilized system. This was further confirmed with the molecular docking results which revealed that CRB is located in the active site residues Try149, Glu152, Ser191, Arg194, Arg198, Trp213, Arg217, Arg256, His287, Ala290, Glu291, Ser343, Asp450 within a radius of 6 Å. Combining the molecular docking studies and the computed thermodynamic parameters, it can be inferred that hydrophobic and electrostatic interactions are the major binding forces involved in formation of the CRB-BSA complex.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

MODELING OF METAL BINDING ON HUMIC SUBSTANCES USING THE NIST DATABASE: AN A PRIORI FUNCTIONAL GROUP APPROACH

EPA Science Inventory

Various modeling approaches have been developed for metal binding on humic substances. However, most of these models are still curve-fitting exercises-- the resulting set of parameters such as affinity constants (or the distribution of them) is found to depend on pH, ionic stren...

Structural basis for bifunctional zinc(II) macrocyclic complex recognition of thymine bulges in DNA.

PubMed

del Mundo, Imee Marie A; Siters, Kevin E; Fountain, Matthew A; Morrow, Janet R

2012-05-07

The zinc(II) complex of 1-(4-quinoylyl)methyl-1,4,7,10-tetraazacyclododecane (cy4q) binds selectively to thymine bulges in DNA and to a uracil bulge in RNA. Binding constants are in the low-micromolar range for thymine bulges in the stems of hairpins, for a thymine bulge in a DNA duplex, and for a uracil bulge in an RNA hairpin. Binding studies of Zn(cy4q) to a series of hairpins containing thymine bulges with different flanking bases showed that the complex had a moderate selectivity for thymine bulges with neighboring purines. The dissociation constants of the most strongly bound Zn(cy4q)-DNA thymine bulge adducts were 100-fold tighter than similar sequences with fully complementary stems or than bulges containing cytosine, guanine, or adenine. In order to probe the role of the pendent group, three additional zinc(II) complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with aromatic pendent groups were studied for binding to DNA including 1-(2-quinolyl)methyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)methyl-1,4,7,10-tetraazacyclododecane (cybp), and 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-N,N-dimethylnaphthalen-1-amine (dsc). The Zn(cybp) complex binds with moderate affinity but little selectivity to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly, Zn(dsc) binds weakly both to thymine bulges and hairpins with fully complementary stems. The zinc(II) complex of cy2q has the 2-quinolyl moiety bound to the Zn(II) center, as shown by (1)H NMR spectroscopy and pH-potentiometric titrations. As a consequence, only weak (500 μM) binding is observed to DNA with no appreciable selectivity. An NMR structure of a thymine-bulge-containing hairpin shows that the thymine is extrahelical but rotated toward the major groove. NMR data for Zn(cy4q) bound to DNA containing a thymine bulge is consistent with binding of the zinc(II) complex to the thymine N3(-) and stacking of the quinoline on top of the thymine. The thymine-bulge bound zinc(II) complex is pointed into the major groove, and there are interactions with the guanine positioned 5' to the thymine bulge.

Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.

PubMed

Anema, Skelte G; de Kruif, C G Kees

2013-07-24

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ∼50 to 100 nm. The κ-casein content decreased markedly and the αS-casein/β-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/κ-casein ratio was constant for all micelle sizes, whereas the lactoferrin/αS-casein and lactoferrin/β-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/αS-casein and lysozyme/β-casein ratios were nearly constant, whereas the lysozyme/κ-casein ratio increased with increasing micelle size, indicating that lysozyme bound to αS-casein and β-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged αS-casein and β-casein.

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

PubMed Central

Alder, J Tracy; Hacksell, Uli; Strange, Philip G

2003-01-01

Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

Quantitative analyses of bifunctional molecules.

PubMed

Braun, Patrick D; Wandless, Thomas J

2004-05-11

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.

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.

Studies on interaction of an intramolecular charge transfer fluorescence probe: 4'-dimethylamino-2,5-dihydroxychalcone with DNA.

PubMed

Xu, Zhicheng; Bai, Guan; Dong, Chuan

2005-10-15

The interaction of a new intramolecular charge transfer probe, namely 4'-dimethylamino-2,5-dihydroxychalcone (DMADHC), with calf thymus DNA has been studied. Compared to the spectral characteristics of the free form in aqueous solution, the fluorescence of DMADHC enhanced dramatically accompanying a blueshift of the emission maxima in the presence of DNA. The absorption and fluorescence spectra, salt concentration effect, KI quenching, fluorescence polarization, and DNA denaturation experiments were given. These results give evidence that the DMADHC molecule is inserted into the base-stacking domain of the DNA double helix. The intrinsic binding constant and the binding site number were estimated. The analytical characteristics were also given.

Ferrocene-Boronic Acid-Fructose Binding Based on Dual-Plate Generator-Collector Voltammetry and Square-Wave Voltammetry.

PubMed

Li, Meng; Xu, Su-Ying; Gross, Andrew J; Hammond, Jules L; Estrela, Pedro; Weber, James; Lacina, Karel; James, Tony D; Marken, Frank

2015-06-10

The interaction of ferrocene-boronic acid with fructose is investigated in aqueous 0.1 m phosphate buffer at pH 7, 8 and 9. Two voltammetric methods, based on 1) a dual-plate generator-collector micro-trench electrode (steady state) and 2) a square-wave voltammetry (transient) method, are applied and compared in terms of mechanistic resolution. A combination of experimental data is employed to obtain new insights into the binding rates and the cumulative binding constants for both the reduced ferrocene-boronic acid (pH dependent and weakly binding) and for the oxidised ferrocene-boronic acid (pH independent and strongly binding).

Long-term bioconcentration kinetics of hydrophobic chemicals in Selenastrum capricornutum and Microcystis aeruginosa

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

Koelmans, A.A.; Woude, H. van der; Hattink, J.

1999-06-01

The bioconcentration of two chlorobenzenes (CBs) and of seven polychlorobiphenyls (PCBs) to Selenastrum capricornutum and Microcystis aeruginosa was studied with accumulation experiments followed by gas purge elimination experiments. Henry's law constants at 10 C were needed to interpret the gas purge results and were measured in control experiments. For the M. aerogunisa culture, steady-state uptake was reached within days, whereas uptake by S. capricornutum took several weeks. The relationships between the log bioconcentration factors (BCF) and log octanol-water partition coefficients (K[sub OW]) were nonlinear, with relatively low values for the more hydrophobic PCBs. Rate constants for the elimination of CBsmore » and PCBs from the algal cells were shown to be larger than 1 per day when calculated with a one-compartment model. With such large rate constants, it is unlikely that the curvature observed for these species is caused by slow kinetics or that algal growth affects BCF by dilution of CB or PCB concentrations. The log BCF-log K[sub OW] relationships could be described by a simple three-phase model that accounted for the binding of CBs and PCBs to dissolved organic carbon (DOC). Modeling bioconcentration of hydrophobic chemicals in phytoplankton should account for the binding to DOC.« less

The interaction of flavonoid-lysozyme and the relationship between molecular structure of flavonoids and their binding activity to lysozyme.

PubMed

Yang, Ran; Yu, Lanlan; Zeng, Huajin; Liang, Ruiling; Chen, Xiaolan; Qu, Lingbo

2012-11-01

In this work, the interactions of twelve structurally different flavonoids with Lysozyme (Lys) were studied by fluorescence quenching method. The interaction mechanism and binding properties were investigated. It was found that the binding capacities of flavonoids to Lys were highly depend on the number and position of hydrogen, the kind and position of glycosyl. To explore the selectivity of the bindings of flavonoids with Lys, the structure descriptors of the flavonoids were calculated under QSAR software package of Cerius2, the quantitative relationship between the structures of flavonoids and their binding activities to Lys (QSAR) was performed through genetic function approximation (GFA) regression analysis. The QSAR regression equation was K(A) = 37850.460 + 1630.01Dipole +3038.330HD-171.795MR. (r = 0.858, r(CV)(2) = 0.444, F((11,3)) = 7.48), where K(A) is binding constants, Dipole, HD and MR was dipole moment, number of hydrogen-bond donor and molecular refractivity, respectively. The obtained results make us understand better how the molecular structures influencing their binding to protein which may open up new avenues for the design of the most suitable flavonoids derivatives with structure variants.

Comparison and analysis on the serum-binding characteristics of aspirin-zinc complex and aspirin.

PubMed

Zhang, Hua-Xin; Zhang, Qun; Wang, Hong-Lin; Li, Li-Wei

2017-09-01

This study was designed to compare the protein-binding characteristics of aspirin-zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three-dimensional fluorescence, ultraviolet-visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non-radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA-binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α-helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

Electrostatic contribution to the binding stability of protein-protein complexes.

PubMed

Dong, Feng; Zhou, Huan-Xiang

2006-10-01

To investigate roles of electrostatic interactions in protein binding stability, electrostatic calculations were carried out on a set of 64 mutations over six protein-protein complexes. These mutations alter polar interactions across the interface and were selected for putative dominance of electrostatic contributions to the binding stability. Three protocols of implementing the Poisson-Boltzmann model were tested. In vdW4 the dielectric boundary between the protein low dielectric and the solvent high dielectric is defined as the protein van der Waals surface and the protein dielectric constant is set to 4. In SE4 and SE20, the dielectric boundary is defined as the surface of the protein interior inaccessible to a 1.4-A solvent probe, and the protein dielectric constant is set to 4 and 20, respectively. In line with earlier studies on the barnase-barstar complex, the vdW4 results on the large set of mutations showed the closest agreement with experimental data. The agreement between vdW4 and experiment supports the contention of dominant electrostatic contributions for the mutations, but their differences also suggest van der Waals and hydrophobic contributions. The results presented here will serve as a guide for future refinement in electrostatic calculation and inclusion of nonelectrostatic effects. Proteins 2006. (c) 2006 Wiley-Liss, Inc.