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Sample records for albumin hsa protein

  1. Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA).

    PubMed

    Dong, Qian; Yan, Xinjian; Kilpatrick, Lisa E; Liang, Yuxue; Mirokhin, Yuri A; Roth, Jeri S; Rudnick, Paul A; Stein, Stephen E

    2014-09-01

    This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

  2. A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

    SciTech Connect

    Zhang, Ling; Wang, Lin; Meng, Zhiyun; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Gao, Lei; Zhu, Xiaoxia; Sun, Wenzhong; Li, Jian; Zheng, Ying; Dou, Guifang

    2014-03-07

    Highlights: • E2HSA has an extended half-life and good plasma stability. • E2HSA could improve glucose-dependent insulin secretion. • E2HSA has excellent glucoregulatory effects in vivo. • E2HSA could potentially be used as a new long-acting GLP-1 receptor agonist for type 2 diabetes management. - Abstract: Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

  3. A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys.

    PubMed

    Zhang, Ling; Wang, Lin; Meng, Zhiyun; Gan, Hui; Gu, Ruolan; Wu, Zhuona; Gao, Lei; Zhu, Xiaoxia; Sun, Wenzhong; Li, Jian; Zheng, Ying; Dou, Guifang

    2014-03-01

    Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

  4. Protection against influenza A virus by vaccination with a recombinant fusion protein linking influenza M2e to human serum albumin (HSA).

    PubMed

    Mu, Xupeng; Hu, Kebang; Shen, Mohan; Kong, Ning; Fu, Changhao; Yan, Weiqun; Wei, Anhui

    2016-02-01

    The highly conserved extracellular domain of M2 protein (M2e) of influenza A viruses has limited immunogenicity on its own. Hence, aiming to enhance immunogenicity of M2e protein, optimal approaches remain to be established. In this study, we created recombinant fusion protein vaccines by linking M2e consensus sequence of influenza A viruses with C-terminal domain of human serum albumin (HSA). Then HSA/M2e recombinant fusion protein was studied. Our results showed that HSA/M2e could induce strong anti-M2e specific humoral immune responses in the established mice model. Administration of HSA/M2e with Freund's adjuvant resulted in a higher number of IFN-γ-producing cells compared to HSA/M2e or M2e peptide emulsified in Freund's adjuvant. Furthermore, HSA/M2e was able to reduce viral load in the mice lungs and provide significant protection against lethal challenge with an H1N1 or an H3N2 virus compared to controls. In conclusion, this study has demonstrated a potential vaccine that could provide protection in preventing the threat of influenza outbreak because of rapid variation of the influenza virus.

  5. Spectroscopic analysis and molecular modeling on the interaction of jatrorrhizine with human serum albumin (HSA)

    NASA Astrophysics Data System (ADS)

    Li, Junfen; Li, Jinzeng; Jiao, Yong; Dong, Chuan

    2014-01-01

    In this work, the interaction of jatrorrhizine with human serum albumin (HSA) was studied by means of UV-vis and fluorescence spectra. The intrinsic fluorescence of HSA was quenched by jatrorrhizine, which was rationalized in terms of the static quenching mechanism. The results show that jatrorrhizine can obviously bind to HSA molecules. According to fluorescence quenching calculations, the bimolecular quenching constant (kq), apparent quenching constant (KSV) at different temperatures were obtained. The binding constants K are 4059 L mol-1 and 1438 L mol-1 at 299 K and 304 K respectively, and the number of binding sites n is almost 1. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants (ΔH -12.25 kJ mol-1 and ΔS 28.17 J mol-1 K-1) clearly indicate that the electrostatic force plays a major role in the process. The efficiency of energy transfer and the distance between the donor (HSA) and the acceptor (jatrorrhizine) were calculated as 22.2% and 3.19 nm according to Föster's non-radiative energy transfer theory. In addition, synchronous fluorescence spectroscopy reveals that jatrorrhizine can influence HSA's microstructure. That is, jatrorrhizine is more vicinal to tryptophane (Trp) residue than to tyrosine (Tyr) residue and the damage site is also mainly at Trp residue. Molecular modeling result shows that jatrorrhizine-HSA complex formed not only on the basis of electrostatic forces, but also on the basis of π-π staking and hydrogen bond. The research results will offer a reference for the studies on the biological effects and action mechanism of small molecule with protein.

  6. Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Khorshidi, Aref; Moghadam, Neda Hossinpour

    2013-10-01

    In the present investigation, an attempt has been made to study the interaction of zonisamide (ZNS) with the transport protein, human serum albumin (HSA) employing UV-Vis, fluorometric, circular dichroism (CD) and molecular docking techniques. The results indicated that binding of ZNS to HSA caused strong fluorescence quenching of HSA through static quenching mechanism, hydrogen bonds and van der Waals contacts are the major forces in the stability of protein ZNS complex and the process of the binding of ZNS with HSA was driven by enthalpy (ΔH = -193.442 kJ mol-1). The results of CD and UV-Vis spectroscopy showed that the binding of this drug to HSA induced conformational changes in HSA. Furthermore, the study of molecular docking also indicated that zonisamide could strongly bind to the site I (subdomain IIA) of HSA mainly by hydrophobic interaction and there were hydrogen bond interactions between this drug and HSA, also known as the warfarin binding site.

  7. PEGylated human serum albumin (HSA) nanoparticles: preparation, characterization and quantification of the PEGylation extent

    NASA Astrophysics Data System (ADS)

    Fahrländer, E.; Schelhaas, S.; Jacobs, A. H.; Langer, K.

    2015-04-01

    Modification with poly(ethylene glycol) (PEG) is a widely used method for the prolongation of plasma half-life of colloidal carrier systems such as nanoparticles prepared from human serum albumin (HSA). However, the quantification of the PEGylation extent is still challenging. Moreover, the influence of different PEG derivatives, which are commonly used for nanoparticle conjugation, has not been investigated so far. The objective of the present study is to develop a method for the quantification of PEG and to monitor the influence of diverse PEG reagents on the amount of PEG linked to the surface of HSA nanoparticles. A size exclusion chromatography method with refractive index detection was established which enabled the quantification of unreacted PEG in the supernatant. The achieved results were confirmed using a fluorescent PEG derivative, which was detected by photometry and fluorimetry. Additionally, PEGylated HSA nanoparticles were enzymatically digested and the linked amount of fluorescently active PEG was directly determined. All the analytical methods confirmed that under optimized PEGylation conditions a PEGylation efficiency of up to 0.5 mg PEG per mg nanoparticle could be achieved. Model calculations made a ‘brush’ conformation of the PEG chains on the particle surface very likely. By incubating the nanoparticles with fetal bovine serum the reduced adsorption of serum proteins on PEGylated HSA nanoparticles compared to non-PEGylated HSA nanoparticles was demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Finally, the positive effect of PEGylation on plasma half-life was demonstrated in an in vivo study in mice. Compared to unmodified nanoparticles the PEGylation led to a four times larger plasma half-life.

  8. Evidence that L-Arginine inhibits glycation of human serum albumin (HSA) in vitro

    SciTech Connect

    Servetnick, D.A.; Wiesenfeld, P.L.; Szepesi, B. )

    1990-02-26

    Previous work by Brownlee has shown that glycation of bovine serum albumin can be reduced in the presence of aminoguanidine (AG). Presumably, the guanidinium group on AG interferes with further rearrangement of amadori products to advanced glycosylated end products (AGE). Since L-arginine (ARG) also contains a guanidinium group, its ability to inhibit the formation of AGE products was investigated. HSA was incubated at 37{degrees}C in the presence or absence of glucose; with glucose and fructose; or with sugars in the presence or absence of ARG or AG. A tracer amount of U-{sup 14}C-glucose was added to each tube containing sugars. Protein bound glucose was separated from unreacted glucose by gel filtration. Radioactivity, total protein, fluorescence, and glucose concentration were measured. Preliminary data show enhanced binding of {sup 14}C-glucose to HSA with fructose at all time points. A 30-40% decrease in {sup 14}C-glucose incorporation was observed when ARG or AG as present. ARG and AG were equally effective in inhibiting incorporation of {sup 14}C-glucose. FPLC analysis is in progress to determine the type and degree of HSA crosslinking during the 2 week incubation period.

  9. Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function

    PubMed Central

    Zhang, Michael S.; Houtman, Jon C. D.

    2016-01-01

    Glycerol monolaurate (GML) is a monoglyceride with well characterized anti-microbial properties. Because of these properties, GML is widely used in food, cosmetics, and personal care products and currently being tested as a therapeutic for menstrual associated toxic shock syndrome, superficial wound infections, and HIV transmission. Recently, we have described that GML potently suppresses select T cell receptor (TCR)-induced signaling events, leading to reduced human T cell effector functions. However, how soluble host factors present in the blood and at sites of infection affect GML-mediated human T cell suppression is unknown. In this study, we have characterized how human serum albumin (HSA) affects GML-induced inhibition of human T cells. We found that HSA and other serum albumins bind to 12 carbon acyl side chain of GML at low micromolar affinities and restores the TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane. Additionally, HSA reverses GML mediated inhibition of AKT phosphorylation and partially restores cytokine production in GML treated cells. Our data reveal that HSA, one of the most abundant proteins in the human serum and at sites of infections, potently reverses the suppression of human T cells by GML. This suggests that GML-driven human T cell suppression depends upon the local tissue environment, with albumin concentration being a major determinant of GML function. PMID:27764189

  10. Interface potential sensing from adsorption of human serum albumin (HSA) on carbon nanotube (CNT) monitored by zero current potentiometry for HSA determination.

    PubMed

    Wang, Huan; Wu, Yi; Song, Jun-Feng

    2015-10-15

    In this work, the adsorption of human serum albumin (HSA) on the bare multiwall carbon nanotube (MWNT) was investigated by a new electrochemical method, termed as zero current potentiometry. For this, a MWNT strip was prepared by directly adhering MWNTs on the transparent adhesive tape surface. Moreover, when HSA adsorbed onto MWNT at the MWNT/solution interface, an interface potential Ψ yielded. The interface potential Ψ as the zero current potential Ezcp simply related to it was monitored by zero current potentiometry. The relationship between the zero current potential Ezcp, the HSA concentration and others was established in simple stoichiometric relation. Based on this, both the adsorption of HSA on MWNT and the HSA determination can be studied. For the HSA determination, the theoretic conclusion consisted with experimental results. The zero current potential Ezcp was proportional to the HSA concentration in the range of 2.8 × 10(-8) - 3.4 × 10(-7)M with the limit of detection 2 × 10(-8)M. The linear regression equation was Ezcp/V (vs, SCE) = (0.159 ± 0.01) + (0.358 ± 0.02) × 10(6)CHSA (µM). This determination was fast, high sensitive and good selective.

  11. Interactions between DMPC liposomes and the serum blood proteins HSA and IgG.

    PubMed

    Sabín, Juan; Prieto, Gerardo; Ruso, Juan M; Messina, Paula V; Salgado, Francisco J; Nogueira, Montserrat; Costas, Miguel; Sarmiento, Félix

    2009-02-12

    The interaction between two serum blood proteins, namely human serum albumin (HSA) and human immunoglobulin G (IgG), with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes has been studied in detail using dynamic light scattering, flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility, differential scanning calorimetry (DSC), and surface tension measurements. HSA and IgG interact with liposomes forming molecular aggregates that remain stable at protein concentrations beyond those of total liposome coverage. Both HSA and IgG penetrate into the liposome bilayer. An ELISA assay indicates that the Fc region of IgG is the one that is immersed in the DMPC membrane. The liposome-protein interaction is mainly of electrostatic nature, but an important hydrophobic contribution is also present.

  12. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction.

    PubMed

    Jupin, Marc; Michiels, Paul J; Girard, Frederic C; Spraul, Manfred; Wijmenga, Sybren S

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  13. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction

    NASA Astrophysics Data System (ADS)

    Jupin, Marc; Michiels, Paul J.; Girard, Frederic C.; Spraul, Manfred; Wijmenga, Sybren S.

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (˜60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  14. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  15. Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSA NPs) alter α-synuclein conformation and aggregation kinetics

    NASA Astrophysics Data System (ADS)

    Mohammad-Beigi, Hossein; Shojaosadati, Seyed Abbas; Marvian, Amir Tayaranian; Pedersen, Jannik Nedergaard; Klausen, Lasse Hyldgaard; Christiansen, Gunna; Pedersen, Jan Skov; Dong, Mingdong; Morshedi, Dina; Otzen, Daniel E.

    2015-11-01

    The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different

  16. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  17. Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: an evaluation of PPIX aggregation in protein-protein interactions.

    PubMed

    Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

    2012-11-01

    Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

  18. Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.

    2015-02-01

    Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur. The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman's reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200-250 nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue. Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was

  19. The effect of albumin fusion patterns on the production and bioactivity of the somatostatin-14 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Yang, Runlin; Peng, Ying; Deng, Lili; Wu, Yu; Fu, Qiang

    2013-08-01

    Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)2-human serum albumin (HSA), (SS14)3-HSA, and HSA-(SS14)3, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)3-HSA and HSA-(SS14)3 was much lower than (SS14)2-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)2-HSA being the most effective one. On the whole, (SS14)2-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity. PMID:23712794

  20. Investigation of the effects of Zn2+, Ca2+ and Na+ ions on the interaction between zonisamide and human serum albumin (HSA) by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Khorshidi, Aref; Mohammadpour, Mahnaz

    2014-03-01

    In this work, The effect of three metal ions Zn2+, Ca2+ and Na1+ on the interaction between human serum albumin (HSA) and zonisamide (ZNS) was investigated employing fluorescence, ultraviolet-visible (UV-Vis) absorption and circular dichroism (CD) under simulated physiological conditions. Fluorescence spectroscopy revealed that these metallic ions and ZNS can quench the HSA fluorescence, and this quenching effect became more significant when both ion and drug are present together. It was found that the quenching mechanism is a combination of static quenching with nonradiative energy transfer. The binding sites number (n), the binding constant (Kb) and the spatial-distance (r) of ZNS with HSA without or with Zn2+, Ca2+ and Na1+ ions were calculated. The presence of Ca2+ and Na+ ions decreased the binding constants (Kb) and the number of binding sites (n) of ZNS-HSA complex. However, the presence of Zn2+ increased the affinity of ZNS for HSA largely. Changes in UV-Vis absorption and CD data are due to the microenvironment of amide moieties in HSA molecules.

  1. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins.

    PubMed

    Tunç, Sibel; Cetinkaya, Ahmet; Duman, Osman

    2013-03-01

    The interactions of tramadol hydrochloride (THC) and 5-azacytidine (AZA) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by fluorescence, UV absorption and circular dichroism (CD) spectroscopy at pH 7.4 and different temperatures. The UV absorption spectra and the fluorescence quenching of HSA and HMG proteins indicated the formation of HSA-THC and HMG-THC complexes via static quenching mechanism. AZA did not interact with HSA and HMG proteins. It was found that the formation of HMG-THC complex was stronger than that of HSA-THC complex. The stability of HSA-THC and HMG-THC complexes decreased with increasing temperature. The number of binding site was found as one for HSA-THC and HMG-THC systems. Negative enthalpy change (ΔH) and Gibbs free energy change (ΔG) and positive entropy change (ΔS) values were obtained for these systems. The binding of THC-HSA and HMG proteins was spontaneous and exothermic. In addition, electrostatic interactions between protein and drug molecules played an important role in the binding processes. The results of CD analysis revealed that the addition of THC led to a significant conformational change in the secondary structure of HSA protein, on the contrary to HMG protein. PMID:23428887

  2. The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Peng, Ying; Yang, Runlin; Deng, Lili; Fu, Qiang

    2014-06-01

    Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.

  3. The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Peng, Ying; Yang, Runlin; Deng, Lili; Fu, Qiang

    2014-06-01

    Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)(2)-HSA, (SS28)(3)-HSA, and HSA-(SS28)(2), were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)(3)-HSA was much lower than (SS28)(2)-HSA and HSA-(SS28)(2) due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)(2)-HSA being the most effective one. A pharmacokinetics study showed that (SS28)(2)-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity. PMID:24752560

  4. Molecular interactions between some non-steroidal anti-inflammatory drugs (NSAID's) and bovine (BSA) or human (HSA) serum albumin estimated by means of isothermal titration calorimetry (ITC) and frontal analysis capillary electrophoresis (FA/CE).

    PubMed

    Ràfols, Clara; Zarza, Sílvia; Bosch, Elisabeth

    2014-12-01

    The interactions between some non-steroidal anti-inflammatory drugs, NSAIDs, (naproxen, ibuprofen and flurbiprofen) and bovine (BSA) or human (HSA) serum albumin have been examined by means of two complementary techniques, isothermal titration calorimetry (ITC) and frontal analysis/capillary electrophoresis (FA/CE). It can be concluded that ITC is able to measure with high precision the strongest drug-albumin interactions but the higher order interactions can be better determined by means of FA/CE. Then, the combination of both techniques leads to a complete evaluation of the binding profiles between the selected NSAIDs and both kind of albumin proteins. When BSA is the binding protein, the NSAIDs show a strong primary interaction (binding constants: 1.5 × 10(7), 8 × 10(5) and 2 × 10(6) M(-1) for naproxen, ibuprofen and flurbiprofen, respectively), and also lower affinity interactions of the same order for the three anti-inflammatories (about 1.7 × 10(4) M(-1)). By contrast, when HSA is the binding protein two consecutive interactions can be observed by ITC for naproxen (9 × 10(5) and 7 × 10(4) M(-1)) and flurbiprofen (5 × 10(6) and 6 × 10(4) M(-1)) whereas only one is shown for ibuprofen (9 × 10(5) M(-1)). Measurements by FA/CE show a single interaction for each drug being the ones of naproxen and flurbiprofen the same that those evaluated by ITC as the second interaction events. Then, the ability of both techniques as suitable complementary tools to establish the whole interaction NSAIDs-albumin profile is experimentally demonstrated and allows foreseeing suitable strategies to establish the complete drug-protein binding profile. In addition, for the interactions analyzed by means of ITC, the thermodynamic signature is established and the relative contributions of the enthalpic and entropic terms discussed.

  5. Exploring the interaction between picoplatin and human serum albumin: The effects on protein structure and activity.

    PubMed

    Wang, Yanqing; Wu, Peirong; Zhou, Xinchun; Zhang, Hongmei; Qiu, Ligan; Cao, Jian

    2016-09-01

    For the first time, the effects of picoplatin on the structure and esterase-like catalytic activity of human serum albumin (HSA) have been investigated by spectroscopic approaches and molecular modeling. The circular dichroism (CD) spectral examinations indicated that the binding of picoplatin with HSA induced a slight decrease of a-helix content of protein and unfolded the constituent polypeptides of the protein. The synchronous fluorescence and three-dimensional fluorescence spectral methods were used to estimate the effect of picoplatin on the micro-environmental changes of the Trp and Tyr residues of HSA, indicating that the micro-environment around the Tyr and Trp residue is partly disturbed by picoplatin. UV-vis absorption spectral result indicated the formation of the ground state complex between picoplatin with HSA. The ANS binding assay indicated the existence of competitive combination of picoplatin and ANS with HSA. The studies on the effects of picoplatin on the binding of HSA with bilirubin and heme showed that picoplatin binding caused a change of angle between two chromophores of bound bilirubin and the binding site of picoplatin does not locate in subdomain IB in HSA that bound with heme. The molecular modeling results showed that picoplatin binds to the connection between domain I and domain II by hydrophobic, hydrogen bonds, and van der Waals forces. In addition, HSA maintains most of its esterase activity in the presence of picoplatin. The investigations on how picoplatin interacts with HSA are important for the understanding of the anticancer mechanism and toxicity of platinum-based anticancer drug. PMID:27484966

  6. Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA

    PubMed Central

    Zhuang, Jianqin; Yang, De-Ping; Tian, Xiaoyu; Nikas, Spyros P.; Sharma, Rishi; Guo, Jason Jianxin; Makriyannis, Alexandros

    2013-01-01

    Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using 19F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain. PMID:24533425

  7. Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary alpha-amylase (HSA) by fluorescence quenching.

    PubMed

    Soares, Susana; Mateus, Nuno; Freitas, Victor de

    2007-08-01

    Phenolic compounds are responsible for major organoleptic characteristics of plant-derived food and beverages; these substances have received much attention, given that the major function of these compounds is their antioxidant ability. In the context of this study, our major aim was study the binding of several phenolic compounds such as (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, malvidin-3-glucoside, tannic acid, procyanidin B4, procyanidin B2 gallate, and procyanidin oligomers to different proteins (bovine serum albumin and human alpha-amylase) by fluorescence quenching of protein intrinsic fluorescence. From the spectra obtained, the Stern-Volmer, the apparent static, and the bimolecular quenching constants were calculated. The structure of polyphenols revealed to significantly affect the binding/quenching process; in general, the binding affinity increased with the molecular weight of polyphenol compounds and in the presence of galloyl groups. For catechin monomer and procyanidin dimer B4, the K(SV) was 14,100 and 13,800 M(-1), respectively, and for galloyl derivatives, the K(SV) was 19,500 and 21,900 M(-1), respectively. Tannic acid was shown to be the major quenching molecule for both proteins. However, comparing different proteins, the same polyphenol showed different quenching effects, which are suggested to be related to the three-dimensional structure of the proteins studied. For (+)-catechin and BSA, the K(SV) was 8700 M(-1), and with alpha-amylase, it was 14,100 M(-1); for tannic acid, the K(SV) was 10,0548 and 11,0674 M(-1), respectively. From the results obtained, besides the main binding analysis performed, we conclude that this technique is more sensitive than thought because we can detect several interactions that have not been proven by other methods, namely, nephelometry. Overall, fluorescence quenching has proven to be a very sensitive technique with many potentialities to analyze the interaction between polyphenols and proteins.

  8. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

    PubMed Central

    Lim, Sung In; Hahn, Young S.; Kwon, Inchan

    2015-01-01

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety. PMID:25862515

  9. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.

    PubMed

    Lim, Sung In; Hahn, Young S; Kwon, Inchan

    2015-06-10

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.

  10. Total Protein and Albumin/Globulin Ratio Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Total Protein and Albumin/Globulin (A/G) Ratio Share this ... Globulin Ratio; A/G Ratio Formal name: Total Protein; Albumin to Globulin Ratio Related tests: Albumin ; Liver ...

  11. Ultrafast dynamics of C30 in solution and within CDs and HSA protein.

    PubMed

    Martin, Cristina; Cohen, Boiko; Gaamoussi, Issam; Ijjaali, Mustapha; Douhal, Abderrazzak

    2014-05-29

    Steady-state UV-visible absorption and emission together with femto to nanosecond time-resolved emission techniques have been applied to study the dynamics of 3-(2-N-methylbenzimidazolyl)-7-(N,N-diethylamino)coumarin (C30) in neat solvents, as well as in the presence of chemical (β-CD and DM-β-CD) and biological (HSA protein) cavities. The formation of inclusion complexes inside the hydrophobic CDs gives 1:1 and 1:2 guest:host complexes, whereas with the HSA protein, the formed 1:1 inclusion complexes are more robust. The picosecond experiments show the importance of the interactions of C30 with the medium, as well as the intramolecular events in the excited-state relaxation as evidenced by the increase in the global emission lifetime from ∼0.5 ns in MeOH/H2O mixtures to 2.5 ns in THF, and to 1-3 ns when the dye is trapped within CDs and HSA cavities. Time-resolved anisotropy (r(t)) results indicate the involvement of ultrafast depolarization processes, whereas in the MeOH/H2O mixtures r(0) = 0.27, in DM-β-CD, r(0) = 0.35. The rotational time decays clearly show the robustness of the formed complexes with CDs and HSA protein: ∼170 ps in MeOH/H2O solvent mixtures, ∼850 ps due to 1:1 and 1:2 β-CD complexes, and 28 ns for HSA complexes. The femtosecond time-resolved emission experiments reveal the significant changes of the dynamics with the encapsulation of C30 by CDs (from approximately τ1 = 0.3 and τ2 = 2 ps in THF to approximately τ1 = 1.0 and τ2 = 7.5 ps in the MeOH/H2O binary mixture, and to approximately τ1 = 3 and τ2 = 30 ps in the CD complexes). The change is explained in terms of how the water molecules modulate the intramolecular charge transfer (ICT) time (τ1) and how the restriction of the environment modifies the torsional process (τ2). In the case of trapped C30 within the HSA protein the intermolecular interactions with the amino acid residues are revealed, giving rise to a complex photodynamical behavior due to the hydrophobic, H

  12. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  13. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. PMID:26821345

  14. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  15. Detailed scrutiny of the anion receptor pocket in subdomain IIA of serum proteins toward individual response to specific ligands: HSA-pocket resembles flexible biological slide-wrench unlike BSA.

    PubMed

    Datta, Shubhashis; Halder, Mintu

    2014-06-12

    Present study reveals that the subdomain IIA cavity of two homologous serum albumins (HSA, BSA) has inherent mutual structural and functional deviations which render noticeable difference in behavior toward specific ligands. The major drug binding site (subdomain IIA) of HSA is found to be largely hydrophobic while that of BSA is partially exposed to water. Larger shift in REE spectra and greater change in solvent reorganization energy of coumarin 343 (C343)-anion in HSA clearly reveals that binding pocket is relatively large and water molecules penetrate deeper into it unlike BSA. The individual response of proteins to perturbation by ligands is found to be way different. Although the subdomain IIA is primarily anion receptive (prefers anionic ligands), the present study suggests that HSA may also like to bind neutral guests due to its remarkable conformational features. Actually, HSA is capable of adopting favorable conformation like mechanical slide-wrench, when required, to accommodate neutral ligands [e.g., coumarin 314 (C314)], as well. But due to less flexible solution structure, BSA behaves like fixed mechanical spanners and hence is not very responsive to C314. Therefore, the generally speaking functional-structural similarities of homologous proteins can be apparent and needs to be analyzed exhaustively.

  16. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research.

  17. Arabidopsis Hsa32, a Novel Heat Shock Protein, Is Essential for Acquired Thermotolerance during Long Recovery after Acclimation1[W

    PubMed Central

    Charng, Yee-yung; Liu, Hsiang-chin; Liu, Nai-yu; Hsu, Fu-chiun; Ko, Swee-suak

    2006-01-01

    Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (>24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants. PMID:16500991

  18. 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.

  19. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  20. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  1. Synthesis, characterization and interaction studies of 1,3,4-oxadiazole derivatives of fatty acid with human serum albumin (HSA): A combined multi-spectroscopic and molecular docking study.

    PubMed

    Laskar, Khairujjaman; Alam, Parvez; Khan, Rizwan Hasan; Rauf, Abdul

    2016-10-21

    An efficient synthesis of fatty acid derivatives of 1,3,4-oxadiazole has been reported and comparative study between conventional heating to that of microwave irradiation also described. The newly synthesized compounds (2A-F) were characterized by FT-IR, (1)H NMR, (13)C NMR and mass spectral analysis. The binding interaction of (Z)-2-(heptadec-8'-enyl)-5-methyl-1,3,4-oxadiazole (2C) with human serum albumin (HSA) has been evaluated by UV, fluorescence, circular dichroism (CD) and molecular docking studies. Fluorescence results showed that compound 2C interacts with HSA through static quenching mechanism with binding affinity of 2 × 10(3) M(-1) and Gibbs free energy change (ΔG) was found to be -16.83 kJ mol(-1). Molecular docking studies have been performed to evaluate possible mode of interaction of compound 2C with HSA.

  2. 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. PMID:23471625

  3. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry.

  4. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  5. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  6. [Binding mechanism of traditional Chinese medicine active component 5-hydroxymethyl-furfural and HSA or BSA].

    PubMed

    Guo, Ming; He, Ling; Lu, Xiao-Wang

    2012-03-01

    A combination of spectral experiment and molecular modeling techniques has been used to characterize the binding mechanism between an active component 5-hydroxymethyl-furfural (5-HMF) of traditional Chinese medicine and human serum albumin (HSA) or bovine serum albumin (BSA). The interaction mechanism of 5-HMF binding with HSA/BSA is analyzed. Although the drug can bind with HSA/BSA to form stable complexes, there are some differences in the bond strength. The values of binding distances (r) are different and low, which indicated the occurrence of energy transfer. The drug had conformational effect on HSA/BSA, which resulted in different changes of hydrophobic environment of the binding domain in HSA/BSA. The 'phase diagram' of fluorescence revealed that the changes on the conformational pattern of proteins have been affected by drug conformed to the "all-or-none" pattern. The interactions between drug and protein influenced by Co(II) were also discussed. Its effects acting on 5-HMF-HSA/BSA interactions are different. The computational modeling method was used to study the interaction between 5-HMF and HSA/BSA. The results of molecular model studies revealed that the binding modes for drug-serum albumin systems are mainly hydrophobic interactions and hydrogen bonding. These results are in accordance with spectral results. The research results have given a better theoretical reference for the study of pharmacological mechanism of 5-hydroxymethyl-furfural.

  7. Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

    PubMed

    Reid, Lara O; Roman, Ernesto A; Thomas, Andrés H; Dántola, M Laura

    2016-08-30

    Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin. PMID:27500308

  8. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins.

  9. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  10. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  11. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  12. Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis.

    PubMed

    Szkudlarek, A; Sułkowska, A; Maciążek-Jurczyk, M; Chudzik, M; Równicka-Zubik, J

    2016-01-01

    Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors --glycation of HSA--occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA(GLC)) with HSA glycated by fructose (gHSA(FRC)). We focused on presenting the differences between gHSA(FRC) and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335nm and λem 420nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSA(FRC) is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSA(FRC) becomes less accessible for the negatively charged quencher (I(-)), KSV value is smaller for gHSA(FRC) than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the

  13. Comparative examination of adsorption of serum proteins on HSA- and PLGA-based nanoparticles using SDS-PAGE and LC-MS.

    PubMed

    Gossmann, R; Fahrländer, E; Hummel, M; Mulac, D; Brockmeyer, J; Langer, K

    2015-06-01

    The behavior of nanosized drug carrier systems under cell culture conditions and therefore also the destiny in the body are highly influenced by the protein corona, which is formed upon entering a biological environment. Some of the adsorbed proteins, named opsonins, lead to a shortened plasma circulation half-life of the nanoparticles. Others are attributed to promote the transport of nanoparticles into other compartments of the body, just to mention two examples. Hence, detailed knowledge concerning the composition of the protein corona is of great importance. The aim of this work was to investigate the influence of the nanoparticle starting material and the surface modification on the composition of the adsorbed serum proteins in a cell culture environment. Therefore, positively charged nanoparticles based on the biodegradable polymer poly(dl-lactide-co-glycolide) (PLGA) stabilized with didodecyldimethylammonium bromide (DMAB) and negatively charged nanoparticles based on human serum albumin (HSA) were prepared and modified with hydrophilic polymers. By incubating the nanoparticles with fetal bovine serum (FBS) the adsorption of serum proteins on the colloidal system was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a semi-quantitative analysis of the protein corona was performed and after enzymatic in-solution-digestion the adsorbed proteins were identified using high resolution LC-MS. Our study accentuates the influence of the core material, surface charge, and surface modification on the amount and nature of the adsorbed proteins. The combination of SDS-PAGE and LC-MS turns out to be a simple and reliable method to investigate the protein corona of nanoparticles.

  14. Combination of UV-vis spectroscopy and chemometrics to understand protein-nanomaterial conjugate: a case study on human serum albumin and gold nanoparticles.

    PubMed

    Wang, Yong; Ni, Yongnian

    2014-02-01

    Study of the interactions between proteins and nanomaterials is of great importance for understanding of protein nanoconjugate. In this work, we choose human serum albumin (HSA) and citrate-capped gold nanoparticles (AuNPs) as a model of protein and nanomaterial, and combine UV-vis spectroscopy with multivariate curve resolution by an alternating least squares (MCR-ALS) algorithm to present a new and efficient method for comparatively comprehensive study of evolution of protein nanoconjugate. UV-vis spectroscopy coupled with MCR-ALS allows qualitative and quantitative extraction of the distribution diagrams, spectra and kinetic profiles of absorbing pure species (AuNPs and AuNPs-HSA conjugate are herein identified) and undetectable species (HSA) from spectral data. The response profiles recovered are converted into the desired thermodynamic, kinetic and structural parameters describing the protein nanoconjugate evolution. Analysis of these parameters for the system gives evidence that HSA molecules are very likely to be attached to AuNPs surface predominantly as a flat monolayer to form a stable AuNPs-HSA conjugate with a core-shell structure, and the binding process takes place mainly through electrostatic and hydrogen-bond interactions between the positively amino acid residues of HSA and the negatively carboxyl group of citrate on AuNPs surface. The results obtained are verified by transmission electron microscopy, zeta potential, circular dichroism spectroscopy and Fourier transform infrared spectroscopy, showing the potential of UV-vis spectroscopy for study of evolution of protein nanoconjugate. In parallel, concentration evolutions of pure species resolved by MCR-ALS are used to construct a sensitive spectroscopic biosensor for HSA with a linear range from 1.8 nM to 28.1 nM and a detection limit of 0.8 nM.

  15. Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

    NASA Astrophysics Data System (ADS)

    Vlasova, I. M.; Vlasov, A. A.; Saletskii, A. M.

    2016-07-01

    The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA-detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.

  16. Superior serum half life of albumin tagged TNF ligands

    SciTech Connect

    Mueller, Nicole; Schneider, Britta; Pfizenmaier, Klaus; Wajant, Harald

    2010-06-11

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

  17. Interaction of Citrinin with Human Serum Albumin.

    PubMed

    Poór, Miklós; Lemli, Beáta; Bálint, Mónika; Hetényi, Csaba; Sali, Nikolett; Kőszegi, Tamás; Kunsági-Máté, Sándor

    2015-12-01

    Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow's Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.

  18. Interaction of Citrinin with Human Serum Albumin

    PubMed Central

    Poór, Miklós; Lemli, Beáta; Bálint, Mónika; Hetényi, Csaba; Sali, Nikolett; Kőszegi, Tamás; Kunsági-Máté, Sándor

    2015-01-01

    Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow’s Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions. PMID:26633504

  19. A retrospective analysis of 25% human serum albumin supplementation in hypoalbuminemic dogs with septic peritonitis

    PubMed Central

    Horowitz, Farrah B.; Read, Robyn L.; Powell, Lisa L.

    2015-01-01

    This study describes the influence of 25% human serum albumin (HSA) supplementation on serum albumin level, total protein (TP), colloid osmotic pressure (COP), hospital stay, and survival in dogs with septic peritonitis. Records of 39 dogs with septic peritonitis were evaluated. In the HSA group, initial and post-transfusion TP, albumin, COP, and HSA dose were recorded. In the non-supplemented group, repeated values of TP, albumin, and COP were recorded over their hospitalization. Eighteen dogs survived (53.8% mortality). Repeat albumin values were higher in survivors (mean 23.9 g/L) and elevated repeat albumin values were associated with HSA supplementation. Repeat albumin and TP were higher in the HSA supplemented group (mean 24 g/L and 51.9 g/L, respectively) and their COP increased by 5.8 mmHg. Length of hospitalization was not affected. Twenty-five percent HSA increases albumin, TP, and COP in canine patients with septic peritonitis. Higher postoperative albumin levels are associated with survival. PMID:26028681

  20. The Relationship between Albumin-Binding Capacity of Recombinant Polypeptide and Changes in the Structure of Albumin-Binding Domain.

    PubMed

    Bormotova, E A; Gupalova, T V

    2015-07-01

    Many bacteria express surface proteins interacting with human serum albumin (HSA). One of these proteins, PAB from anaerobic bacteria, contains an albumin-binding domain consisting of 45 amino acid residues known as GA domain. GA domains are also found in G proteins isolated from human streptococcal strains (groups C and G) and of albumin-binding protein isolated from group G streptococcal strains of animal origin. The GA domain is a left-handed three-helix bundle structure in which amino acid residues of the second and third helixes are involved in albumin binding. We studied the relationship between HSA-binding activity of the recombinant polypeptide isolated from group G streptococcus of animal origin and structure of the GA domain is studied. Structural changes in GA domain significantly attenuated HAS-binding capacity of the recombinant polypeptide. Hence, affinity HSA-binding polypeptide depends on stability of GA domain structure.

  1. KINETIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY USING PEAK PROFILING AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: EXAMINATION OF MULTI-SITE INTERACTIONS OF DRUGS WITH HUMAN SERUM ALBUMIN COLUMNS

    PubMed Central

    Tong, Zenghan; Schiel, John E.; Papastavros, Efthimia; Ohnmacht, Corey M.; Smith, Quentin R.; Hage, David S.

    2010-01-01

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (± 0.2) s-1 and 0.67 (± 0.04) s-1 at pH 7.4 and 37 °C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins. PMID:21067755

  2. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  3. Interaction of coffee compounds with serum albumins. Part II: Diterpenes.

    PubMed

    Guercia, Elena; Forzato, Cristina; Navarini, Luciano; Berti, Federico

    2016-05-15

    Cafestol and 16-O-methylcafestol are diterpenes present in coffee, but whilst cafestol is found in both Coffea canephora and Coffea arabica, 16-O-methylcafestol (16-OMC) was reported to be specific of only C. canephora. The interactions of such compounds, with serum albumins, have been studied. Three albumins have been considered, namely human serum albumin (HSA), fatty acid free HSA (ffHSA) and bovine serum albumin (BSA). The proteins interact with the diterpenes at the interface between Sudlow site I and the fatty acid binding site 6 in a very peculiar way, leading to a significant change in the secondary structure. The diterpenes do not displace reference binding drugs of site 2, but rather they enhance the affinity of the site for the drugs. They, therefore, may alter the pharmacokinetic profile of albumin - bound drugs.

  4. Imatinib binding to human serum albumin modulates heme association and reactivity.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; Trezza, Viviana; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2014-10-15

    Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, α1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to HSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 °C and 37.0 °C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo. PMID:25057771

  5. Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

    PubMed

    Maly, Jan; Stanek, Ondrej; Frolik, Jan; Maly, Marek; Ennen, Franka; Appelhans, Dietmar; Semeradtova, Alena; Wrobel, Dominika; Stofik, Marcel; Knapova, Tereza; Kuchar, Milan; Stastna, Lucie Cervenkova; Cermak, Jan; Sebo, Peter; Maly, Petr

    2016-04-01

    For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

  6. Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

    PubMed

    Maly, Jan; Stanek, Ondrej; Frolik, Jan; Maly, Marek; Ennen, Franka; Appelhans, Dietmar; Semeradtova, Alena; Wrobel, Dominika; Stofik, Marcel; Knapova, Tereza; Kuchar, Milan; Stastna, Lucie Cervenkova; Cermak, Jan; Sebo, Peter; Maly, Petr

    2016-04-01

    For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures. PMID:26748571

  7. About the structural role of disulfide bridges in serum albumins: evidence from protein simulated unfolding.

    PubMed

    Paris, Guillaume; Kraszewski, Sebastian; Ramseyer, Christophe; Enescu, Mironel

    2012-11-01

    The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues.

  8. 2S Albumin Storage Proteins: What Makes them Food Allergens?

    PubMed Central

    Moreno, F. Javier; Clemente, Alfonso

    2008-01-01

    2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response. PMID:18949071

  9. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

  10. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. PMID:24316526

  11. Interaction of prodigiosin with HSA and β-Lg: Spectroscopic and molecular docking studies.

    PubMed

    Rastegari, Banafsheh; Karbalaei-Heidari, Hamid Reza; Yousefi, Reza; Zeinali, Sedigheh; Nabavizadeh, Masoud

    2016-04-01

    Human serum albumin (HSA) and bovine β-lactoglobulin (β-Lg) are both introduced as blood and oral carrier scaffolds with high affinity for a wide range of pharmaceutical compounds. Prodigiosin, a natural three pyrrolic compound produced by Serratia marcescens, exhibits many pharmaceutical properties associated with health benefits. In the present study, the interaction of prodigiosin with HSA and β-Lg was investigated using fluorescence spectroscopy, circular dichroism (CD) and computational docking. Prodigiosin interacts with the Sudlow's site I of HSA and the calyx of β-Lg with association constant of 4.41 × 10(4) and 1.99 × 10(4) M(-1) to form 1:1 and 2:3 complexes at 300K, respectively. The results indicated that binding of prodigiosin to HSA and β-Lg caused strong fluorescence quenching of both proteins through static quenching mechanism. Electrostatic and hydrophobic interactions are the major forces in the stability of PG-HSA complex with enthalpy- and entropy-driving mode, although the formation of prodigiosin-β-Lg complex is entropy-driven hydrophobic associations. CD spectra showed slight conformational changes in both proteins due to the binding of prodigiosin. Moreover, the ligand displacement assay, pH-dependent interaction and protein-ligand docking study confirmed that the prodigiosin binds to residues located in the subdomain IIA and IIIA of HSA and central calyx of β-Lg. PMID:26924214

  12. Interaction of prodigiosin with HSA and β-Lg: Spectroscopic and molecular docking studies.

    PubMed

    Rastegari, Banafsheh; Karbalaei-Heidari, Hamid Reza; Yousefi, Reza; Zeinali, Sedigheh; Nabavizadeh, Masoud

    2016-04-01

    Human serum albumin (HSA) and bovine β-lactoglobulin (β-Lg) are both introduced as blood and oral carrier scaffolds with high affinity for a wide range of pharmaceutical compounds. Prodigiosin, a natural three pyrrolic compound produced by Serratia marcescens, exhibits many pharmaceutical properties associated with health benefits. In the present study, the interaction of prodigiosin with HSA and β-Lg was investigated using fluorescence spectroscopy, circular dichroism (CD) and computational docking. Prodigiosin interacts with the Sudlow's site I of HSA and the calyx of β-Lg with association constant of 4.41 × 10(4) and 1.99 × 10(4) M(-1) to form 1:1 and 2:3 complexes at 300K, respectively. The results indicated that binding of prodigiosin to HSA and β-Lg caused strong fluorescence quenching of both proteins through static quenching mechanism. Electrostatic and hydrophobic interactions are the major forces in the stability of PG-HSA complex with enthalpy- and entropy-driving mode, although the formation of prodigiosin-β-Lg complex is entropy-driven hydrophobic associations. CD spectra showed slight conformational changes in both proteins due to the binding of prodigiosin. Moreover, the ligand displacement assay, pH-dependent interaction and protein-ligand docking study confirmed that the prodigiosin binds to residues located in the subdomain IIA and IIIA of HSA and central calyx of β-Lg.

  13. Determination of the structure and morphology of gold nanoparticle-HSA protein complexes.

    PubMed

    Capomaccio, Robin; Jimenez, Isaac Ojea; Colpo, Pascal; Gilliland, Douglas; Ceccone, Giacomo; Rossi, François; Calzolai, Luigi

    2015-11-14

    We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems. PMID:26462441

  14. Binding of Sulpiride to Seric Albumins.

    PubMed

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

    2016-01-04

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

  15. Binding of Sulpiride to Seric Albumins

    PubMed Central

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

    2016-01-01

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

  16. Binding of Sulpiride to Seric Albumins.

    PubMed

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

    2016-01-01

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

  17. Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

    NASA Astrophysics Data System (ADS)

    Liu, Jinxiang; Deng, Qiliang; Tao, Dingyin; Yang, Kaiguang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-06-01

    Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g-1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

  18. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  19. Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters

    PubMed Central

    Yamada, Kana; Yokomaku, Kyoko; Haruki, Risa; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2016-01-01

    A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior. PMID:26895315

  20. Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters.

    PubMed

    Yamada, Kana; Yokomaku, Kyoko; Haruki, Risa; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2016-01-01

    A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

  1. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  2. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  3. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  4. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urinary protein or albumin (nonquantitative) test... Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a) Identification. A urinary protein or albumin (nonquantitative) test system is a device intended to...

  5. The Sialic Acid Binding Protein, Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species

    PubMed Central

    Zhou, Peng; Liu, Jinman; Li, Xiaoli; Takahashi, Yukihiro; Qi, Fengxia

    2015-01-01

    Dental biofilm development involves initial colonization of the tooth’s surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S. gordonii also is a pathogen in infective endocarditis in susceptible humans. A surface adhesin, Hsa, has been shown to play a critical role in colonization of S. gordonii on the heart tissue; however, its role in oral biofilm development has not been reported. In this study we demonstrate that Hsa is essential for coaggregation between S. gordonii and Veillonella sp., which are bridging species connecting the pioneer colonizers to the late colonizers. Interestingly, the same domains shown to be required for Hsa binding to sialic acid on the human cell surface are also required for coaggregation with Veillonella sp. However, sialic acid appeared not to be required for this intergeneric coaggregation. This result suggests that although the same domains of Hsa are involved in binding to eukaryotic as well as Veillonella cells, the binding mechanism is different. The gene expression pattern of hsa was also studied and shown not to be induced by coaggregation with Veillonella sp. PMID:26606595

  6. The Sialic Acid Binding Protein, Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species.

    PubMed

    Zhou, Peng; Liu, Jinman; Li, Xiaoli; Takahashi, Yukihiro; Qi, Fengxia

    2015-01-01

    Dental biofilm development involves initial colonization of the tooth's surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S. gordonii also is a pathogen in infective endocarditis in susceptible humans. A surface adhesin, Hsa, has been shown to play a critical role in colonization of S. gordonii on the heart tissue; however, its role in oral biofilm development has not been reported. In this study we demonstrate that Hsa is essential for coaggregation between S. gordonii and Veillonella sp., which are bridging species connecting the pioneer colonizers to the late colonizers. Interestingly, the same domains shown to be required for Hsa binding to sialic acid on the human cell surface are also required for coaggregation with Veillonella sp. However, sialic acid appeared not to be required for this intergeneric coaggregation. This result suggests that although the same domains of Hsa are involved in binding to eukaryotic as well as Veillonella cells, the binding mechanism is different. The gene expression pattern of hsa was also studied and shown not to be induced by coaggregation with Veillonella sp. PMID:26606595

  7. Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

  8. Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

    NASA Astrophysics Data System (ADS)

    Szkudlarek, A.; Sułkowska, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.

    2016-01-01

    Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors - glycation of HSA - occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSAGLC) with HSA glycated by fructose (gHSAFRC). We focused on presenting the differences between gHSAFRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335 nm and λem 420 nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSAFRC is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSAFRC becomes less accessible for the negatively charged quencher (I-), KSV value is smaller for gHSAFRC than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum

  9. Structural basis of non-steroidal anti-inflammatory drug diclofenac binding to human serum albumin.

    PubMed

    Zhang, Yao; Lee, Philbert; Liang, Shichu; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2015-11-01

    Human serum albumin (HSA) is the most abundant protein in plasma, which plays a central role in drug pharmacokinetics because most compounds bound to HSA in blood circulation. To understand binding characterization of non-steroidal anti-inflammatory drugs to HSA, we resolved the structure of diclofenac and HSA complex by X-ray crystallography. HSA-palmitic acid-diclofenac structure reveals two distinct binding sites for three diclofenac in HSA. One diclofenac is located at the IB subdomain, and its carboxylate group projects toward polar environment, forming hydrogen bond with one water molecule. The other two diclofenac molecules cobind in big hydrophobic cavity of the IIA subdomain without interactive association. Among them, one binds in main chamber of big hydrophobic cavity, and its carboxylate group forms hydrogen bonds with Lys199 and Arg218, as well as one water molecule, whereas another diclofenac binds in side chamber, its carboxylate group projects out cavity, forming hydrogen bond with Ser480.

  10. Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor.

    PubMed

    Li, Liangliang; Guo, Qiang; Liu, Ju; Zhang, Jun; Yin, Ying; Dong, Dayong; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-01-01

    Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs. PMID:26805881

  11. Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor.

    PubMed

    Li, Liangliang; Guo, Qiang; Liu, Ju; Zhang, Jun; Yin, Ying; Dong, Dayong; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-01-20

    Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs.

  12. Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

    PubMed

    Gerlza, Tanja; Winkler, Sophie; Atlic, Aid; Zankl, Christina; Konya, Viktoria; Kitic, Nikola; Strutzmann, Elisabeth; Knebl, Kerstin; Adage, Tiziana; Heinemann, Akos; Weis, Roland; Kungl, Andreas J

    2015-08-01

    Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber

  13. Hemoglobin–Albumin Cluster Incorporating a Pt Nanoparticle: Artificial O2 Carrier with Antioxidant Activities

    PubMed Central

    Hosaka, Hitomi; Haruki, Risa; Yamada, Kana; Böttcher, Christoph; Komatsu, Teruyuki

    2014-01-01

    A covalent core–shell structured protein cluster composed of hemoglobin (Hb) at the center and human serum albumins (HSA) at the periphery, Hb-HSAm, is an artificial O2 carrier that can function as a red blood cell substitute. Here we described the preparation of a novel Hb-HSA3 cluster with antioxidant activities and its O2 complex stable in aqueous H2O2 solution. We used an approach of incorporating a Pt nanoparticle (PtNP) into the exterior HSA unit of the cluster. A citrate reduced PtNP (1.8 nm diameter) was bound tightly within the cleft of free HSA with a binding constant (K) of 1.1×107 M−1, generating a stable HSA-PtNP complex. This platinated protein showed high catalytic activities for dismutations of superoxide radical anions (O2•–) and hydrogen peroxide (H2O2), i.e., superoxide dismutase and catalase activities. Also, Hb-HSA3 captured PtNP into the external albumin unit (K = 1.1×107 M−1), yielding an Hb-HSA3(PtNP) cluster. The association of PtNP caused no alteration of the protein surface net charge and O2 binding affinity. The peripheral HSA-PtNP shell prevents oxidation of the core Hb, which enables the formation of an extremely stable O2 complex, even in H2O2 solution. PMID:25310133

  14. Methylated DNA Binding Domain Protein 2 (MBD2) Coordinately Silences Gene Expression through Activation of the MicroRNA hsa-mir-496 Promoter in Breast Cancer Cell Line

    PubMed Central

    Alvarado, Sebastian; Wyglinski, Joanne; Suderman, Matthew; Andrews, Stephen A.; Szyf, Moshe

    2013-01-01

    Methylated DNA binding protein 2 (MBD2) binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA) antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets. PMID:24204564

  15. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  16. 4-alkoxyethoxy-N-octadecyl-1,8-naphthalimide fluorescent sensor for human serum albumin and other major blood proteins: design, synthesis and solvent effect.

    PubMed

    Wei, Song; Sun, Yang; Tan, Chunlei; Yan, Sen; Guo, Ping; Hu, Xiaoyun; Fan, Jun

    2013-01-01

    A series of 4-alkoxyethoxy-N-octadecyl-1,8-naphthalimides with intense blue fluorescence were designed and synthesized as polarity and spectrofluorimetric probes for the determination of proteins. In solvents of different polarities, the Stokes shifts of two dyes increased with increasing solvent polarity and fluorescence quantum yields decreased significantly, suggesting that electronic transiting from ground to excited states was π-π(*) in character. Dipole moment changes were estimated from solvent-dependent Stokes shift data using a solvatochromic method based on bulk solvent polarity functions and the microscopic solvent polarity parameter (E(T)N). These results were generally consistent with semi-empirical molecular orbital calculations and were found to be quite reliable based on the fact that the correlation of the solvatochromic Stokes shifts with E(T)N was superior to that obtained using bulk solvent polarity functions. Fluorescence data revealed that the fluorescence quenching of human serum albumin (HSA) by dyes was the result of the formation of a Dye-HSA complex. The method was applied to the determination of total proteins (HSA + immunoglobulins) in human serum samples and results were in good agreement with those reported by the research institute.

  17. Amadori albumin in diabetic nephropathy

    PubMed Central

    Neelofar, Km.; Ahmad, Jamal

    2015-01-01

    Nonenzymatic glycation of macromolecules in diabetes mellitus (DM) is accelerated due to persistent hyperglycemia. Reducing sugar such as glucose reacts non enzymatically with free €-amino groups of proteins through series of reactions forming Schiff bases. These bases are converted into Amadori product and further into AGEs. Non enzymatic glycation has the potential to alter the biological, structural and functional properties of macromolecules both in vitro and in vivo. Studies have suggested that amadori as well as AGEs are involved in the micro-macro vascular complications in DM, but most studies have focused on the role of AGEs in vascular complications of diabetes. Recently putative AGE-induced patho-physiology has shifted attention from the possible role of amadori-modified proteins, the predominant form of the glycated proteins in the development of the diabetic complications. Human serum albumin (HSA), the most abundant protein in circulation contains 59 lysine and 23 arginine residues that could, in theory be involved in glycation. Albumin has dual nature, first as a marker of intermediate glycation and second as a causative agent of the damage of tissues. Among the blood proteins, hemoglobin and albumin are the most common proteins that are glycated. HSA with a shorter half life than RBC, appears to be an alternative marker of glycemic control as it can indicate blood glucose status over a short period (2-3 weeks) and being unaffected by RBCs life span and variant haemoglobin, anemia etc which however, affect HbA1c. On the other hand, Amadori albumin may accumulate in the body tissues of the diabetic patients and participate in secondary complications. Amadori-albumin has potential role in diabetic glomerulosclerosis due to long term hyperglycaemia and plays an important role in the pathogenesis of diabetic nephropathy. This review is an approach to compile both the nature of glycated albumin as a damaging agent of tissues and as an intermediate

  18. Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Mertz, Damien; Affolter-Zbaraszczuk, Christine; Barthès, Julien; Cui, Jiwei; Caruso, Frank; Baumert, Thomas F.; Voegel, Jean-Claude; Ogier, Joelle; Meyer, Florent

    2014-09-01

    In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing. Electronic supplementary information (ESI) available: Experimental details and supporting Fig. S1-S4. See DOI: 10.1039/c4nr02623c

  19. Analysis of drug-protein interactions by high-performance affinity chromatography: interactions of sulfonylurea drugs with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Anguizola, Jeanethe; Hoy, Krina S; Hage, David S

    2015-01-01

    High-performance affinity chromatography (HPAC) is a type of liquid chromatography that has seen growing use as a tool for the study of drug-protein interactions. This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug-protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experiments will be presented, as well as factors to consider in the extension of these methods to other drugs and proteins or additional types of biological interactions. PMID:25749961

  20. Photo selective protein immobilization using bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Kim, Wan-Joong; Kim, Ansoon; Huh, Chul; Park, Chan Woo; Ah, Chil Seong; Kim, Bong Kyu; Yang, Jong-Heon; Chung, Kwang Hyo; Choi, Yo Han; Hong, Jongcheol; Sung, Gun Yong

    2012-11-01

    A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

  1. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    PubMed

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

  2. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    PubMed

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression. PMID:26668316

  3. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

    PubMed

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-04-11

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

  4. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    SciTech Connect

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  5. Spectral Fluorescence Properties of an Anionic Oxacarbocyanine Dye in Complexes with Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Pronkin, P. G.; Tatikolov, A. S.

    2015-07-01

    The spectral fluorescence properties of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC) were studied in solutions and in complexes with human serum albumin (HSA). Interaction with HSA leads to a significant increase in the fluorescence of the dye. We studied quenching of the fluorescence of OCC in a complex with HSA by ibuprofen and warfarin. Data on quenching of fluorescence by ibuprofen indicate binding of the dye to binding site II of subdomain IIIA in the HSA molecule. Synchronous fluorescence spectra of human serum albumin in the presence of OCC showed that complexation with OCC does not lead to appreciable rearrangement of the protein molecule at the binding site.

  6. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice*

    PubMed Central

    Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

    2013-01-01

    Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism. PMID:24101203

  7. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.

    PubMed

    Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

    2013-10-01

    Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.

  8. The effect of albumin on podocytes: The role of the fatty acid moiety and the potential role of CD36 scavenger receptor

    SciTech Connect

    Pawluczyk, I.Z.A.; Pervez, A.; Ghaderi Najafabadi, M.; Saleem, M.A.; Topham, P.S.

    2014-08-15

    Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA{sub +FA}) or depleted of them (HSA{sub −FA}). Receptor-mediated endocytosis of FITC-HSA{sub +FA} over 60 min was 5 times greater than that of FITC-HSA{sub −FA}. 24 h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA{sub −FA.} Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin. - Highlights: • The fatty acid moiety is essential for receptor mediated endocytosis of albumin. • Fatty acid depleted albumin is more pathogenic to podocytes. • CD36 is not directly involved in albumin uptake by podocytes.

  9. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    PubMed Central

    Akbarzadehlaleh, Parvin; Mirzaei, Mona; Mashahdi-Keshtiban, Mahdiyeh; Shamsasenjan, Karim; Heydari, Hamidreza

    2016-01-01

    Human serum albumin (HSA) is a non-glycosylated, negatively charged protein (Mw: about 65-kDa) that has one free cystein residue (Cys 34), and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days). As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP) in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG), that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase), and compares various methods to purifiy and characterize the pegylated form. PMID:27766215

  10. Recombinant albumin monolayers on latex particles.

    PubMed

    Sofińska, Kamila; Adamczyk, Zbigniew; Kujda, Marta; Nattich-Rak, Małgorzata

    2014-01-14

    The adsorption of recombinant human serum albumin (rHSA) on negatively charged polystyrene latex micro-particles was studied at pH 3.5 and the NaCl concentration range of 10(-3) to 0.15 M. The electrophoretic mobility of latex monotonically increased with the albumin concentration in the suspension. The coverage of adsorbed albumin was quantitatively determined using the depletion method, where the residual protein concentration was determined by electrokinetic measurements and AFM imaging. It was shown that albumin adsorption was irreversible. Its maximum coverage on latex varied between 0.7 mg m(-2) for 10(-3) M NaCl to 1.3 mg m(-2) for 0.15 M NaCl. The latter value matches the maximum coverage previously determined for human serum albumin on mica using the streaming potential method. The increase in the maximum coverage was interpreted in terms of reduced electrostatic repulsion among adsorbed molecules. These facts confirm that albumin adsorption at pH 3.5 is governed by electrostatic interactions and proceeds analogously to colloid particle deposition. The stability of albumin monolayers was measured in additional experiments where changes in the latex electrophoretic mobility and the concentration of free albumin in solutions were monitored over prolonged time periods. Based on these experimental data, a robust procedure of preparing albumin monolayers on latex particles of well-controlled coverage and molecule distribution was proposed. PMID:24354916

  11. Cu(II) Bis(thiosemicarbazone) Radiopharmaceutical Binding to Serum Albumin: Further Definition of Species-Dependence and Associated Substituent Effects

    PubMed Central

    Basken, Nathan E.; Green, Mark A.

    2009-01-01

    Introduction The Cu-PTSM (pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N4-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) radiopharmaceutical appears to only exhibit non-specific binding to human and animal serum albumins. Methods To further probe the structural basis for the species-dependence of this albumin binding interaction, protein binding of these three radiopharmaceuticals was examined in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat, elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species-dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate. PMID:19520290

  12. Inhalable self-assembled albumin nanoparticles for treating drug-resistant lung cancer.

    PubMed

    Choi, Seong Ho; Byeon, Hyeong Jun; Choi, Ji Su; Thao, Lequang; Kim, Insoo; Lee, Eun Seong; Shin, Beom Soo; Lee, Kang Choon; Youn, Yu Seok

    2015-01-10

    Direct pulmonary delivery of anti-cancer agents is viewed as an effective way of treating lung cancer. Here, we fabricated inhalable nanoparticles made of human serum albumin (HSA) conjugated with doxorubicin and octyl aldehyde and adsorbed with apoptotic TRAIL protein (TRAIL/Dox HSA-NP). The octyl aldehyde and doxorubicin endowed HSA with significant hydrophobicity that facilitated self-assembly. TRAIL/Dox HSA-NP was found to have excellent particle size (~340nm), morphology, dispersability, and aerosolization properties. TRAIL/Dox HSA-NP displayed synergistic cytotoxicity and apoptotic activity in H226 lung cancer cells vs. HSA-NP containing TRAIL or Dox alone. TRAIL/Dox HSA-NP was well deposited in the mouse lungs using an aerosolizer, and TRAIL and Dox-HSA were found to be gradually released over 3days. The anti-tumor efficacy of pulmonary administered TRAIL/Dox HSA-NP was evaluated in BALB/c nu/nu mice bearing H226 cell-induced metastatic tumors. It was found that the tumors of H226-implanted mice treated with TRAIL/Dox HSA-NP were remarkably smaller and lighter than those of mice treated with TRAIL or Dox HSA-NP alone (337.5±7.5; 678.2±51.5; and 598.9±24.8mg, respectively). Importantly, this improved anti-tumor efficacy was found to be due to the synergistic apoptotic effects of Dox and TRAIL. In the authors' opinion, TRAIL/Dox HSA-NP offers a potential inhalable anti-lung cancer drug delivery system. Furthermore, the synergism displayed by combined use of Dox and TRAIL could be used to markedly reduce doxorubicin doses and minimize its side effects.

  13. Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

    PubMed

    Zhuang, Shulin; Wang, Haifei; Ding, Keke; Wang, Jiaying; Pan, Liumeng; Lu, Yanli; Liu, Qingjun; Zhang, Chunlong

    2016-02-01

    Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials.

  14. Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

    PubMed

    Zhuang, Shulin; Wang, Haifei; Ding, Keke; Wang, Jiaying; Pan, Liumeng; Lu, Yanli; Liu, Qingjun; Zhang, Chunlong

    2016-02-01

    Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials. PMID:26454115

  15. Impact of albumin on drug delivery--new applications on the horizon.

    PubMed

    Elsadek, Bakheet; Kratz, Felix

    2012-01-10

    Over the past decades, albumin has emerged as a versatile carrier for therapeutic and diagnostic agents, primarily for diagnosing and treating diabetes, cancer, rheumatoid arthritis and infectious diseases. Market approved products include fatty acid derivatives of human insulin or the glucagon-like-1 peptide (Levemir(®) and Victoza(®)) for treating diabetes, the taxol albumin nanoparticle Abraxane(®) for treating metastatic breast cancer which is also under clinical investigation in further tumor indications, and (99m)Tc-aggregated albumin (Nanocoll(®) and Albures(®)) for diagnosing cancer and rheumatoid arthritis as well as for lymphoscintigraphy. In addition, an increasing number of albumin-based or albumin-binding drugs are in clinical trials such as antibody fusion proteins (MM-111) for treating HER2/neu positive breast cancer (phase I), a camelid albumin-binding nanobody anti-HSA-anti-TNF-α (ATN-103) in phase II studies for treating rheumatoid arthritis, an antidiabetic Exendin-4 analog bound to recombinant human albumin (phase I/II), a fluorescein-labeled albumin conjugate (AFL)-human serum albumin for visualizing the malignant borders of brain tumors for improved surgical resection, and finally an albumin-binding prodrug of doxorubicin (INNO-206) entering phase II studies against sarcoma and gastric cancer. In the preclinical setting, novel approaches include attaching peptides with high-affinity for albumin to antibody fragments, the exploitation of albumin-binding gadolinium contrast agents for magnetic resonance imaging, and physical or covalent attachment of antiviral, antibacterial, and anticancer drugs to albumin that are permanently or transiently attached to human serum albumin (HSA) or act as albumin-binding prodrugs. This review gives an overview of the expanding field of preclinical and clinical drug applications and developments that use albumin as a protein carrier to improve the pharmacokinetic profile of the drug or to target the drug

  16. Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy.

    PubMed

    Liu, Yu-Kuo; Li, Yu-Teng; Lu, Ching-Fan; Huang, Li-Fen

    2015-05-25

    Fusion of the sugar-starvation-induced αAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L(-1) in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem. PMID:25765580

  17. Human serum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis.

    PubMed

    Byeon, Hyeong Jun; Min, Sun Young; Kim, Insoo; Lee, Eun Seong; Oh, Kyung Taek; Shin, Beom Soo; Lee, Kang Choon; Youn, Yu Seok

    2014-12-17

    Albumin conjugation is viewed as an effective means of protracting short in vivo lifespans of proteins and targeting rheumatoid arthritis (RA). In this study, we present a human serum albumin (HSA) conjugate linked with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a bifunctional PEG derivative (HSA-TRAIL). Prepared HSA-TRAIL was found to have a larger molecular size (∼240 kDa, 15.4 nm) than TRAIL (∼66 kDa, 6.2 nm), and its bioactivity (apoptosis, cytotoxicity, and antiproliferation) was well preserved in Mia Paca-2 cells and mouse splenocytes. The enhanced therapeutic efficacy of HSA-TRAIL was demonstrated in collagen-induced arthritis (CIA) mice. The incidence and clinical scores, expressed as degree of erythema and swelling in HSA-TRAIL-treated mice, were remarkably lower than those of TRAIL-treated mice. The serum levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, and IL-2 in HSA-TRAIL-treated mice were significantly lower than those of TRAIL-treated mice. Furthermore, HSA-TRAIL accumulated in the hind paws of CIA mice, not in naïve TRAIL mice. Pharmacokinetic profiles of HSA-TRAIL were greatly improved in comparison to those of TRAIL (AUCinf: 844.1 ± 130.0 vs 36.0 ± 1.2 ng·h/mL; t1/2: 6.20 ± 0.72 vs 0.23 ± 0.01 h, respectively). The HSA-TRAIL conjugate, which presents clear advantages of targeting RA and long systemic circulation by HSA and unique anti-inflammatory efficacy by TRAIL, has potential as a novel treatment for rheumatoid arthritis. PMID:25387356

  18. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

    PubMed

    Sandhya, B; Hegde, Ashwini H; Kalanur, Shankara S; Katrahalli, Umesha; Seetharamappa, J

    2011-04-01

    The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Förster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.

  19. Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinaris

    SciTech Connect

    Gupta, Pankaj; Gaur, Vineet; Salunke, Dinakar M.

    2008-08-01

    A 2S albumin from L. culinaris was purified and crystallized and preliminary crystallographic studies were carried out. Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5 kDa) has been identified using NH{sub 2}-terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH{sub 2}-terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5 Å resolution and were indexed in space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 78.6, c = 135.2 Å.

  20. New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

    PubMed Central

    Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Fülöp, Gábor Á.; Csató, Viktória; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Szentkirályi, István Elek; Maros, Tamás Miklós; Szerafin, Tamás; Édes, István; Papp, Zoltán; Tóth, Attila

    2014-01-01

    About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC50) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo. PMID:24691203

  1. Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles.

    PubMed

    Selva Sharma, Arumugam; Ilanchelian, Malaichamy

    2015-07-30

    In the present investigation, we have systematically studied the binding mechanism of model protein human serum albumin (HSA) with gold/silver alloy nanoparticles (Au/Ag NPs) using multiple spectroscopic techniques. Absorption spectral studies of Au/Ag NPs in the presence of increasing concentrations of HSA resulted in a slight red shift of the surface plasmon resonance band (SPR) of Au/Ag NPs, suggesting changes in the refractive index around the nanoparticle surface owing to the adsorption of HSA. The results from high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta potential analysis substantiated the formation of a dense layer of HSA on the surface of Au/Ag NPs. The formation of a ground-state complex between HSA and Au/Ag NPs was evident from the outcome of the steady-state emission titration experiments of the HSA-Au/Ag NPs system. The binding parameters computed from corrected emission quenching data revealed that HSA exhibited a significant binding affinity toward Au/Ag NPs. The identical fluorescence lifetime values of HSA and HSA-Au/Ag NPs from time-resolved fluorescence spectroscopic analysis further authenticated the findings of steady-state emission measurements. The formation of HSA corona on the Au/Ag NPs surface was established on the basis of experimental quenching data and theoretical values. The occurrence of partial unfolding of HSA upon its interaction with the Au/Ag NPs surface was established by using an extrinsic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS). Absorption, Fourier transform infrared (FT-IR), Raman, circular dichroism (CD), and excitation-emission matrix (3D) spectral studies were also carried out to explore Au/Ag NPs-induced tertiary and secondary conformational changes of HSA. The influence of Au/Ag NPs on the esterase-like activity of HSA was established by probing the hydrolysis of p-nitrophenyl acetate. PMID:26106942

  2. Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin).

    PubMed

    Sheng, Yao; Qi, Xiaozhe; Liu, Yifei; Guo, Mingzhang; Chen, Siyuan; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2014-10-01

    Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

  3. A study on human serum albumin influence on glycation of fibrinogen

    SciTech Connect

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2013-09-13

    Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [{sup 13}C{sub 6}] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein.

  4. Competitive binding of phenylbutazone and colchicine to serum albumin in multidrug therapy: A spectroscopic study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Maciążek-Jurczyk, M.; Bojko, B.; Równicka, J.; Zubik-Skupień, I.; Temba, E.; Pentak, D.; Sułkowski, W. W.

    2008-06-01

    The binding sites for phenylbutazone and colchicine were identified in tertiary structure of bovine and human serum albumin with the use of spectrofluorescence analysis. It was found that phenylbutazone has two binding sites in both sera albumins (HSA and BSA), while colchicine has one binding site in BSA as well as in HSA. The comparison of the quenching effect of BSA and HSA fluorescence by phenylbutazone and colchicine allows us to identify subdomain IIA in protein as the binding site for these two drugs. In this subdomain tryptophan 214 is located. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-albumin complex. The comparison of quenching of fluorescence of HSA and BSA excited at 280 nm with that at 295 nm allowed us to state that the participation of tyrosyl residues of albumin in the phenylbutazone-serum albumin interaction is significant. The analysis of quenching of fluorescence of BSA in the binary and ternary systems showed that phenylbutazone does not affect the complex formed between colchicine and BSA. Similarly, colchicine has no effect on the Phe-BSA complex. However marked differences were observed for the complex with HSA. On the basis of Ka and KQ values it was concluded that colchicine may probably cause displacement of phenylbutazone from its complex with serum albumin (SA). Static and dynamic quenching for the binary and ternary systems is also discussed. The competition of phenylbutazone and colchicine in binding to serum albumin should be taken into account in the multi-drug therapy.

  5. 21 CFR 862.1645 - Urinary protein or albumin (nonquantitative) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1645 Urinary protein or albumin (nonquantitative) test system. (a... are characterized by proteinuria or albuminuria. (b) Classification. Class I (general controls)....

  6. Probing the Sudlow binding site with warfarin: how does gold nanocluster growth alter human serum albumin?

    PubMed

    Russell, B A; Mulheran, P A; Birch, D J S; Chen, Y

    2016-08-17

    The search for new fluorescent molecules is vital to the advancement of molecular imaging and sensing for the benefit of medical and biological studies. One such class of new fluorescent molecule is fluorescent gold nanoclusters encapsulated in Human Serum Albumin (HSA-AuNC). In order to use this new fluorescent molecule as a sensor or fluorescent marker in biological imaging both in vitro and in vivo it is important to understand whether/how the proteins function is changed by the synthesis and presence of the gold nanoclusters inside the protein. Natural HSA acts as the main drug carrier in the blood stream, carrying a multitude of molecules in two major binding sites (Sudlow I and II). To test the effects of gold on the ability of HSA to act as a drug carrier we employed warfarin, an anticoagulant drug, as a fluorescent probe to detect changes between natural HSA and HSA-AuNCs. AuNCs are found to inhibit the take up of warfarin by HSA. Evidence for this is found from fluorescence spectral and lifetime measurements. Interestingly, the presence of warfarin bound to HSA also inhibits the formation of gold nanoclusters within protein. This research provides valuable insight into how protein function can change upon synthesis of AuNCs and how that will affect their use as a fluorescent probe.

  7. Protein degradation by ruminal microorganisms from sheep fed dietary supplements of urea, casein, or albumin.

    PubMed Central

    Wallace, R J; Broderick, G A; Brammall, M L

    1987-01-01

    Ruminal fluid from sheep fed hay plus concentrate diets containing 1.8% urea, 6% casein, or 6% egg albumin had proteolytic activities of 4.12, 3.02, or 4.00 mg of [14C]casein hydrolyzed ml-1 h-1, respectively. Dietary albumin had no effect on the rate of albumin breakdown relative to that of casein (0.06). Greater numbers of highly proteolytic bacteria, mainly Butyrivibrio spp., were isolated from the rumens of sheep receiving albumin. Albumin hydrolysis by these isolates was even slower relative to that of casein (0.03) than in ruminal fluid and was similar to that found in isolates from urea- and casein-fed sheep. Hence, there appears to be no mechanism by which ruminal bacteria can alter their proteolytic activity to utilize resistant soluble protein more effectively. PMID:3579280

  8. Changes of protein solutions during storage: a study of albumin pharmaceutical preparations.

    PubMed

    Christiansen, Cathrine; Skotland, Tore

    2010-03-05

    During the production of air-filled albumin microspheres, to be used as an ultrasound contrast agent, it was observed that some albumin lots could not be used owing to albumin precipitation. In order to understand the reason for these lot-to-lot variations, 24 lots of 5% (w/v) human albumin pharmaceutical preparations were analysed. The results revealed that the good albumin lots all contained <0.03 mol of free SH groups per mol of albumin. The precipitation observed with other lots was most probably due to higher amounts of free SH groups. The lower amount of free SH groups in the good lots correlated with: (i) a yellow colour of the solutions and a UV-visible spectrum similar to that observed for non-enzymatic glycosylation; (ii) a decreased fructosamine content; (iii) an increased mobility against the anode in isoelectric focusing; and (iv) an increased truncation of the two N-terminal amino acids. No, or only small, differences were observed for the amounts of albumin dimer, albumin aggregates and protein impurities, and these could not account for the albumin precipitation. The differences observed between the albumin lots were most probably due to varying storage times and/or storage conditions, and incubation experiments revealed changes in all parameters that differed between the good and bad lots. Increasing the storage temperature or exposing the solutions to light resulted in a faster decrease of free SH groups and increase of the yellow colouration. It is likely that at least some of the changes observed were due to reactive degradation products formed from the stabilizer N-acetyl-L-tryptophan. The results presented should also be of interest regarding the storage of monoclonal antibodies and other proteins used in pharmaceuticals.

  9. Spectroscopic studies on the interaction between tetrandrine and two serum albumins by chemometrics methods

    NASA Astrophysics Data System (ADS)

    Cheng, Zhengjun; Liu, Rong; jiang, Xiaohui

    2013-11-01

    The binding interactions of tetrandrine (TETD) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by spectroscopic methods. These experimental data were further analyzed using multivariate curve resolution-alternating least squares (MCR-ALS) method, and the concentration profiles and pure spectra for three species (BSA/HSA, TETD and TETD-BSA/HSA) existed in the interaction procedure, as well as, the apparent equilibrium constants Kapp were evaluated. The binding sites number n and the binding constants K were obtained at various temperatures. The binding distance between TETD and BSA/HSA was 1.455/1.451 nm. The site markers competitive experiments indicated that TETD primarily bound to the tryptophan residue of BSA/HSA within site I. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TETD-BSA was mainly depended on the hydrophobic interaction strongly and electrostatic interaction, and yet the binding of TETD-HSA was strongly relied on the hydrophobic interaction. The results of synchronous fluorescence, 3D fluorescence and FT-IR spectra show that the conformation of proteins has altered in the presence of TETD. In addition, the effect of some common ions on the binding constants between TETD and proteins were also discussed.

  10. Co-amoxiclav Effects on the Structural and Binding Properties of Human Serum Albumin

    PubMed Central

    Hesami Takallu, Saeed; Rezaei Tavirani, Mostafa; Kalantari, Shiva; Amir Bakhtiarvand, Mahrooz; Mahdavi, Sayed Mohammad

    2010-01-01

    Human serum albumin (HSA) is the most abundant plasma protein in the human body. HSA plays an important role in drug transport and metabolism. This protein has a high affinity to a very wide range of materials, including metals such as Cu2+ and Zn2+, fatty acids, amino acids and metabolites such as bilirubin and many drug compounds. In this study, we investigated the effects of co-amoxiclav, as a drug which could be carried by this protein, on HSA structure and binding properties via spectroscopy and electrochemistry techniques. Based on this study, it was found that a therapeutic dose of co-amoxiclav as well as doses 4 to 8 folds higher than the therapeutic dose has no considerable effect on the HSA tertiary structure at 37oC. However, a dose 2 folds that of the therapeutic dose has a slight effect, but higher doses of the drug has a mild effect in pathological temperature (42oC). In addition, charge density of HSA surface is decreased at 42oC, compared to 37oC. Hence, this finding suggests a reduced role of HSA in regulation of osmotic pressure in the fever conditions, compared to the physiological conditions. Co-amoxiclav reduces the charge surface density of HSA at physiological and pathological temperatures and therefore alters its binding properties, which could be important in drug interference and complications. PMID:24363734

  11. Probing the binding of an endocrine disrupting compound-Bisphenol F to human serum albumin: Insights into the interactions of harmful chemicals with functional biomacromolecules

    NASA Astrophysics Data System (ADS)

    Pan, Fang; Xu, Tianci; Yang, Lijun; Jiang, Xiaoqing; Zhang, Lei

    2014-11-01

    Bisphenol F (BPF) as an endocrine disrupting compounds (EDCs) pollutant in the environment poses a great threat to human health. To evaluate the toxicity of BPF at the protein level, the effects of BPF on human serum albumin (HSA) were investigated at three temperatures 283, 298, and 308 K by multiple spectroscopic techniques. The experimental results showed that BPF effectively quenched the intrinsic fluorescence of HSA via static quenching. The number of binding sites, the binding constant, the thermodynamic parameters and the binding subdomain were measured, and indicated that BPF could spontaneously bind with HSA on subdomain IIA through H-bond and van der Waals interactions. Furthermore, the conformation of HSA was demonstrably changed in the presence of BPF. The work provides accurate and full basic data for clarifying the binding mechanisms of BPF with HSA in vivo and is helpful for understanding its effect on protein function during its transportation and distribution in blood.

  12. 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.

  13. Influence of diets with different levels of protein and energy on liver albumin content in the rat.

    PubMed

    Maurice, M; Lardeux, B; De Saint-Steban, C; Bourdel, G; Feldmann, G

    1986-11-01

    The influence of protein ingestion on liver albumin synthesis and albumin content was investigated in rats fed protein as a meal (90% casein) given apart from the other dietary components provided ad libitum. In this condition, protein ingestion rapidly stimulates liver total protein synthesis. Separately fed rats were studied 6 and 20 h after the protein meal. Control rats fed mixed diets containing 13 or 80% casein were killed either during the absorptive (night) or postabsorptive (light) periods. The ratio of hepatic albumin synthesis to total protein synthesis remained fairly constant (12-15%) in all groups, indicating that albumin synthesis paralleled total protein synthesis. Liver albumin content measured in microsomes by immunonephelometry was significantly higher in separately fed rats killed 6 h postmeal than in those killed after 20 h. In rats fed 13% casein, the liver albumin content remained high regardless of the time of killing. In rats fed 80% casein, the albumin content was higher during the absorptive period than during the postabsorptive period. Immunoperoxidase staining of the hepatocyte organelles involved in albumin synthesis, especially the Golgi apparatus, was more intense for separately fed rats killed 6 h postmeal than for those killed after 20 h. Livers of rats fed 13% casein also exhibited a pattern indicative of high hepatocyte albumin content, whereas livers of rats fed 80% casein contained less. These results show that, in separate feeding, wide circadian variations of albumin synthesis run parallel to changes in liver albumin content.

  14. The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

    PubMed Central

    Barbosa, Leandro R.S.; Ortore, Maria Grazia; Spinozzi, Francesco; Mariani, Paolo; Bernstorff, Sigrid; Itri, Rosangela

    2010-01-01

    Abstract The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ∼35–45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0–9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects. PMID:20085727

  15. Impact of preoperative serum albumin on 30-day mortality following surgery for colorectal cancer: a population-based cohort study

    PubMed Central

    Montomoli, Jonathan; Erichsen, Rune; Antonsen, Sussie; Nilsson, Tove; Sørensen, Henrik Toft

    2015-01-01

    Objective Surgery is the only potentially curable treatment for colorectal cancer (CRC), but it is hampered by high mortality. Human serum albumin (HSA) below 35 g/L is associated with poor overall prognosis in patients with CRC, but evidence regarding the impact on postoperative mortality is sparse. Methods We performed a population-based cohort study including patients undergoing CRC surgery in North and Central Denmark (1997–2011). We categorised patients according to HSA concentration measured 1–30 days prior to surgery date. We used the Kaplan-Meier method to compute 30-day mortality and Cox regression model to compute HRs as measures of the relative risk of death, controlling for potential confounders. We further stratified patients by preoperative conditions, including cancer stage, comorbidity level, and C reactive protein concentration. Results Of the 9339 patients undergoing first-time CRC surgery with preoperative HSA measurement, 26.4% (n=2464) had HSA below 35 g/L. 30-day mortality increased from 4.9% among patients with HSA 36–40 g/L to 26.9% among patients with HSA equal to or below 25 g/L, compared with 2.0% among patients with HSA above 40 g/L. The corresponding adjusted HRs increased from 1.75 (95% CI 1.25 to 2.45) among patients with HSA 36–40 g/L to 7.59 (95% CI 4.95 to 11.64) among patients with HSA equal to or below 25 g/L, compared with patients with HSA above 40 g/L. The negative impact associated with a decrement of HSA was found in all subgroups. Conclusions A decrement in preoperative HSA concentration was associated with substantial concentration-dependent increased 30-day mortality following CRC surgery. PMID:26462287

  16. Startling temperature effect on proteins when confined: single molecular level behaviour of human serum albumin in a reverse micelle.

    PubMed

    Sengupta, Bhaswati; Yadav, Rajeev; Sen, Pratik

    2016-06-01

    The present work reports the effect of confinement, and temperature therein, on the conformational fluctuation dynamics of domain-I of human serum albumin (HSA) by fluorescence correlation spectroscopy (FCS). The water-pool of a sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelle has been used as the confined environment. It was observed that the conformational fluctuation time is about 6 times smaller compared to bulk medium when confined in a water-pool of 3.5 nm radius. On increasing the size of the water-pool the conformational fluctuation time was found to increase monotonically and approaches the bulk value. The effect of confinement is on par with the general belief about the restricted motion of a macromolecule upon confinement. However, the effect of temperature was found to be surprising. An increase in the temperature from 298 K to 313 K induces a larger change in the conformational fluctuation time in HSA, when confined. In the bulk medium, apparently there is no change in the conformational fluctuation time in the aforementioned temperature range, whereas, when HSA is present in an AOT water-pool of radius 3.5 nm, about an 88% increase in the fluctuation time was observed. The observed prominent thermal effect on the conformational dynamics of domain-I of HSA in the water-pool of an AOT reverse micelle as compared to in the bulk medium was concluded to arise from the confined solvent effect.

  17. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life

    PubMed Central

    Adams, Ralph; Griffin, Laura; Compson, Joanne E.; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair DG.; Humphreys, David P.; Heywood, Sam

    2016-01-01

    ABSTRACT We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1–7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 – pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively. PMID:27315033

  18. Exploring binding properties of sertraline with human serum albumin: Combination of spectroscopic and molecular modeling studies.

    PubMed

    Shahlaei, Mohsen; Rahimi, Behnoosh; Nowroozi, Amin; Ashrafi-Kooshk, Mohammad Reza; Sadrjavadi, Komail; Khodarahmi, Reza

    2015-12-01

    Human serum albumin (HSA)-drug binding is an important factor to determine half life and bioavailability of drugs. In the present research, the interaction of sertraline (SER) to HSA was investigated using combination of spectroscopic and molecular modeling techniques. Changes in the UV-Vis, CD and FT-IR spectra as well as a significant degree of tryptophan fluorescence quenching were observed upon SER-HSA interaction. Data obtained by spectroscopic methods along with the computational studies suggest that SER binds to residues located in subdomain IIA of HSA. Analysis of spectroscopic data represented the formation of 1:1 complex, significant binding affinity, negative values of entropy and enthalpy changes and the essential role of hydrophobic interactions in binding of SER to HSA. The binding models were demonstrated in the aspects of SER's conformation, active site interactions, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of SER confirmed that the ligand to be bound in a large hydrophobic cavity of HSA. In accordance with experimental data, computational analyses indicated that SER binding does not alter the secondary structure of the protein. The results not only lead to a better understanding of interaction between SER and HSA but also provide useful data about the influence of SER on the protein conformation. PMID:26471709

  19. Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques.

    PubMed

    Li, Xiangrong; Yang, Zhenhua

    2015-05-01

    Oridonin has been traditionally and widely used for treatment of various human diseases due to its uniquely biological, pharmacological and physiological functions. In this study, the interaction between oridonin and human serum albumin (HSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy and UV-vis absorption spectroscopy. We found that the hydrogen bond and van der Waals force are the major binding forces in the binding of oridonin to HSA. The binding of oridonin to HSA is driven by favorable enthalpy and unfavorable entropy. Oridonin can quench the fluorescence of HSA through a static quenching mechanism. The binding constant between oridonin and HSA is moderate and the equilibrium fraction of unbound oridonin f(u) > 60%. Binding site I is found to be the primary binding site for oridonin. Additionally, oridonin may induce conformational changes of HSA and affect its biological function as the carrier protein. The results of the current study suggest that oridonin can be stored and transported from the circulatory system to reach its target organ to provide its therapeutic effects. But its side-effect in the clinics cannot be overlook. The study provides an accurate and full basic data for clarifying the binding mechanism of oridonin with HSA and is helpful for understanding its effect on protein function during the blood transportation process and its biological activity in vivo.

  20. Replica exchange Monte Carlo simulation of human serum albumin-catechin complexes.

    PubMed

    Li, Yunqi; An, Lijia; Huang, Qingrong

    2014-09-01

    Replica exchange Monte Carlo simulation equipped with an orientation-enhanced hydrophobic interaction was utilized to study the impacts of molar ratio and ionic strength on the complex formation of human serum albumin (HSA) and catechin. Only a small amount of catechins was found to act as bridges in the formation of HSA-catechin complexes. Selective binding behavior was observed at low catechin to HSA molar ratio (R). Increase of catechin amount can suppress HSA self-aggregation and diminish the selectivity of protein binding sites. Strong saturation binding with short-range interactions was found to level off at around 4.6 catechins per HSA on average, while this number slowly increased with R when long-range interactions were taken into account. Meanwhile, among the three rings of catechin, the 3,4-dihydroxyphenyl (B-ring) shows the strongest preference to bind HSA. Neither the aggregation nor the binding sites of the HSA-catechin complex was sensitive to ionic strength, suggesting that the electrostatic interaction is not a dominant force in such complexes. These results provide a further molecular level understanding of protein-polyphenol binding, and the strategy employed in this work shows a way to bridge phase behaviors at macroscale and the distribution of binding sites at residue level. PMID:25111890

  1. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin: Effects of esteryl position on affinity.

    PubMed

    Tang, Bin; Huang, Yanmei; Ma, Xiangling; Liao, Xiaoxiang; Wang, Qing; Xiong, Xinnuo; Li, Hui

    2016-12-01

    Structural differences among various dietary polyphenols affect their absorption, metabolism, and bioactivities. In this work, chlorogenic acid (CA) and its two positional isomers, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA), were investigated for their binding reactions with human serum albumin (HSA) using fluorescence, ultraviolet-visible, Fourier transform infrared and circular dichroism spectroscopies, as well as molecular docking. All three isomers were bound to HSA at Sudlow's site I and affected the protein secondary structure. CCA presented the strongest ability of hydrogen-bond formation, and both CA and NCA generated more electrostatic interactions with HSA. The albumin-binding capacity of these compounds decreased in the order CCA>NCA>CA. The compound with 4-esteryl structure showed higher binding affinity and larger conformational changes to HSA than that with 3- or 5-esteryl structures. These comparative studies on structure-affinity relationship contributed to the structural modification and design of phenolic food additives or new polyphenol-like drugs. PMID:27374553

  2. Hsa-miR-137, hsa-miR-520e and hsa-miR-590-3p perform crucial roles in Lynch syndrome

    PubMed Central

    Zhou, Changyu; Li, Jiayu; Li, Jiarui; Wan, Yingchun; Li, Tao; Ma, Piyong; Wang, Yingjian; Sang, Haiyan

    2016-01-01

    The aim of the present study was to identify the differentially expressed microRNAs (DEMs) between Lynch syndrome (LS) and the normal colonic (N-C) control samples, predict the target genes (TGs) and analyze the potential functions of the DEMs and TGs. The miRNA expression dataset GSE30454, which included data of 13 LS and 20 N-C tissue samples, was downloaded from the Gene Expression Omnibus. The classical t-test in Linear Models for Microarray Data package was used for DEM identification. TG prediction was performed using 5 databases. The regulatory network of the DEMs and their TGs was constructed using Cytoscape. Functional and pathway enrichment analysis was performed. The transcription factors (TFs), tumor-associated genes (TAG) and tumor suppressor genes (TSGs) were then identified. Three key DEMs hsa-miR-137, hsa-miR-520e, and hsa-miR-590-3p were identified. Hsa-miR-520e and hsa-miR-137 had 4 common TGs, including SNF related kinase, metal-regulatory transcription factor 1 (MTF1), round spermatid basic protein 1 and YTH N6-methyladenosine RNA binding protein 3; hsa-miR-590-3p and hsa-miR-137 had 14 common TGs, including NCK adaptor protein 1 (NCK1), EPH receptor A7, and stress-associated endoplasmic reticulum protein 1; hsa-miR-590-3p and hsa-miR-520e had 12 common TGs, including Krüppel-like factor (KLF) 13, twinfilin actin binding protein 1, and nuclear factor I B. Through the functional and pathway enrichments analysis, MTF1 was involved in regulation of gene expression and metabolic processes, and sequence-specific DNA binding TF activity. KLF13 was involved in regulation of gene expression and regulation of cellular metabolic processes. NCK1 was enriched in the axon guidance pathway. In addition, the functional and pathway enrichment analysis showed certain TGs, such as hypoxia-inducible factor 1α, AKT serine/threonine kinase 2, and rapamycin-insensitive companion of mammalian target of rapamycin, participated in the mTOR signaling pathway. The 3 key

  3. Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

    PubMed

    Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

    2015-12-01

    The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).

  4. Chlorpromazine interactions to sera albumins. A study by the quenching of fluorescence

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Cortez, Célia M.; Louro, Sônia R. W.

    2004-04-01

    Binding of chlorpromazine (CPZ) and hemin (Hmn) to human (HSA) and bovine (BSA) serum albumin was studied by fluorescence quenching technique. Intrinsic fluorescences of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with CPZ and Hmn. CPZ is a widely used anti-psychosis drug that causes severe side effects and strongly interacts with biomembranes, both in its lipidic and proteic regions. CPZ also interacts with blood components, influences bioavailability, and affects the function of several biomolecules. Albumin plays an important role in the transport and storage of hormones, ions, fatty acids and others substances, including CPZ, affecting the regulation of their plasmatic concentration. Hmn is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with great specificity. Hmn added to HSA and BSA solutions at a molar ratio of 1:1 quenched about half of their fluorescence. Stern-Volmer plots obtained from experiments carried out at 25 and 35 °C showed the quenching of fluorescence of HSA and BSA by CPZ to be a collisional phenomenon. Hmn quenches fluorescence by a static process, which specifically indicates the formation of a complex. Our results suggest the prime binding site for CPZ and Hmn on both HSA and BSA to be near tryptophan residues.

  5. Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin

    NASA Astrophysics Data System (ADS)

    Yue, Yuanyuan; Dong, Qiao; Zhang, Yajie; Li, Xiaoge; Yan, Xuyang; Sun, Yahui; Liu, Jianming

    2016-01-01

    Small molecular drugs that can combine with target proteins specifically, and then block relative signal pathway, finally obtain the purpose of treatment. For this reason, the synthesis of novel imidazole derivatives was described and this study explored the details of imidazole derivatives binding to human serum albumin (HSA). The data of steady-state and time-resolved fluorescence showed that the conjugation of imidazole derivatives with HSA yielded quenching by a static mechanism. Meanwhile, the number of binding sites, the binding constants, and the thermodynamic parameters were also measured; the raw data indicated that imidazole derivatives could spontaneously bind with HSA through hydrophobic interactions and hydrogen bonds which agreed well with the results from the molecular modeling study. Competitive binding experiments confirmed the location of binding. Furthermore, alteration of the secondary structure of HSA in the presence of the imidazole derivatives was tested.

  6. Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular β-sheet formation.

    PubMed

    Bhattacharya, Arpan; Prajapati, Roopali; Chatterjee, Surajit; Mukherjee, Tushar Kanti

    2014-12-16

    Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 μM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular β-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between β-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 μM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the

  7. Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography.

    PubMed

    Dong, Yuesheng; Zhang, Fan; Wang, Zhiming; Du, Li; Hao, Aiyu; Jiang, Bo; Tian, Mingyu; Li, Qiao; Jia, Qian; Wang, Shicong; Xiu, Zhilong

    2012-07-01

    Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K₂HPO₄ aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01-73 L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS-PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1-2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61 μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation. PMID:22658659

  8. Effects of Fenton Reaction on Human Serum Albumin: An In Vitro Study

    PubMed Central

    Khosravifarsani, Meysam; Monfared, Ali Shabestani; Pouramir, Mahdi; Zabihi, Ebrahim

    2016-01-01

    Introduction Human serum albumin (HSA) is a critical protein in human blood plasma, which can be highly damaged by oxidative stress. The aim of this study was to analyze modifications of this protein after oxidation using a Fenton system. Methods In this 2015 experiment, different ratios of Fenton reagent (Fe2+/H2O2) was incubated with one concentration of human serum albumin (1mg/ml). Hence, HSA was incubated 30 min with various combinations of a Fenton system and quantified oxidation products such as carbonyl groups, fragmentations, degradations, and oxidized free thiol group using reliable techniques. Image and data analysis were carried out using ImageJ software and Excel (version 2007), respectively. Results An SDS-PAGE profile showed no cross link and aggregation. However, protein band intensity has decreased to 50% in the highest ratio of H2O2/Fe. Carbonylation assay indicated carbonyl/protein (molc/molp) ratio increased linearly in lower ratios and the values plateau at higher levels of H2O2/Fe 2+. The only free sulfhydryl group on HSA was oxidized in all ratios of the Fenton system. Conclusion To sum, the structure of HSA has been changed following treatment with Hydroxyl Radical as the main product of Fenton reaction. These data confirm the antioxidant activity of HSA. PMID:27790352

  9. Hyperglycemia induced structural and functional changes in human serum albumin of diabetic patients: a physico-chemical study.

    PubMed

    Neelofar, Km; Arif, Zarina; Alam, Khursheed; Ahmad, Jamal

    2016-07-19

    Structural and functional changes in albumin are of particular interest as numerous studies in vivo have reported a strong involvement of glycated-HSA in the development and progression of chronic diabetic complications. Non-enzymatic addition of glucose molecules to a protein induces structural changes in it. These changes depend on the degree of glycation. In this study, conformational changes in glycated-HSA and its antioxidant capacity were evaluated. HSA was purified from diabetic patients with/without CKD and healthy subjects. Glycation induced an increase in the molecular mass of HSA as determined by mass spectroscopy. Further secondary and tertiary structural changes were observed by UV, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), tryptophan and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. The mean α-helix content was found to be 59.46% for normal HSA and it was reduced down to 45.63% in HSA isolated from diabetic patients without CKD and to 37.48% in CKD-HSA. FTIR analysis showed Amide I and Amide II band shifting in HSA of diabetic patients without and with CKD. These findings indicate the secondary structure changes in glycated HSA. The tertiary structure is also affected by in vivo glycation as confirmed by intrinsic fluorescence and ANS fluorescence results. Consequently, these structural changes associated with glycation provoked a reduction in the free thiol group and a strong increment of protein carbonyl contents and the fructosamine level in glycated HSA. Antioxidant activity was evaluated by a RBC hemolysis test. The result indicates that the free radical scavenging capacities of HSA were decreased in diabetic patients with or without CKD. Our study revealed that structural and functional features of glycated HSA, isolated from diabetic patients with and without CKD were significantly different from the HSA isolated from non-diabetic subjects. Moreover these changes were more prominent in HSA

  10. Study on the Mechanism of Interaction Between Tubeimoside I and Human Serum Albumin at Different Temperatures by Three-Dimensional Fluorescence Spectrum

    NASA Astrophysics Data System (ADS)

    Lin, Xiaogang; Li, Wenchao; Ye, Changbin; Liu, Zhiyuan

    2015-06-01

    Tubeimoside (TBMS), the bulb of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae), is one of the traditional Chinese medicines often used for the treatment of tumors as well as for detoxication. Tubeimoside I (TBMS I) is one of the main active ingredients of TBMS, the mechanism of action of which remains unknown. Human serum albumin (HSA) is the most abundant carrier protein in blood circulation. Three-dimensional (3D) fluorescence spectra and the excitation-emission matrix of interaction between TBMS I and HSA were measured at different temperatures. The results showed that HSA fluorescence was quenched by TBMS I through a static quenching mechanism. Also, the HSA fluorescence was quenched with the temperature increase from 283 K to 353 K. 3D spectral results revealed the changes in the secondary structure of HSA upon interaction with TBMS I.

  11. Recognition of oxidized albumin and thyroid antigens by psoriasis autoantibodies

    PubMed Central

    Al-Shobaili, Hani A.; Ahmed, Ahmed A.; Rasheed, Zafar

    2015-01-01

    Objectives: To investigate the role of reactive-oxygen-species (ROS) induced epitopes on human-serum-albumin (HSA) and thyroid antigens in psoriasis autoimmunity. Methods: This study was performed in the College of Medicine, Qassim University, Buraidah, Saudi Arabia between May 2014 and February 2015. The study was designed to explore the role of ROS-induced epitopes in psoriasis autoimmunity. Singlet-oxygen (or ROS)-induced epitopes on protein (ROS-epitopes-albumin) was characterized by in-vitro and in-vivo. Thyroid antigens were prepared from rabbit thyroid, and thyroglobulin was isolated from thyroid extract. Immunocross-reactions of protein-A purified anti-ROS-epitopes-HSA-immunoglobulin G (IgGs) with thyroid antigen, thyroglobulin, and their oxidized forms were determined. Binding characteristics of autoantibodies in chronic plaque psoriasis patients (n=26) against ROS-epitopes-HSA and also with native and oxidized thyroid antigens were screened, and the results were compared with age-matched controls (n=22). Results: The anti-ROS-epitopes-HSA-IgGs showed cross-reactions with thyroid antigen, thyroglobulin and with their oxidized forms. High degree of specific binding by psoriasis IgGs to ROS-epitopes-HSA, ROS-thyroid antigen and ROS-thyroglobulin was observed. Immunoglobulin G from normal-human-controls showed negligible binding with all tested antigens. Moreover, sera from psoriasis patients had higher levels of carbonyl contents compared with control sera. Conclusion: Structural alterations in albumin, thyroid antigens by ROS, generate unique neo-epitopes that might be one of the factors for the induction of autoantibodies in psoriasis. PMID:26620982

  12. Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

    PubMed Central

    Loste, A; Marca, M C

    1999-01-01

    The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration. PMID:10369572

  13. Study on the interaction of catechins with human serum albumin using spectroscopic and electrophoretic techniques

    NASA Astrophysics Data System (ADS)

    Trnková, Lucie; Boušová, Iva; Staňková, Veronika; Dršata, Jaroslav

    2011-01-01

    The interaction between eight naturally occurring flavanols (catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate) and human serum albumin (HSA) has been investigated by spectroscopic (fluorescence quenching and UV-Vis absorption) and electrophoretic (native and SDS PAGE) techniques under simulated physiological conditions (pH 7.40, 37 °C). The spectroscopic results confirmed the complex formation for the tested systems. The binding constants and the number of binding sites were obtained by analysis of fluorescence data. The strongest binding affinity to HSA was found for epicatechin gallate and decreased in the order epicatechin gallate ⩾ catechin gallate > epigallocatechin gallate > gallocatechin gallate ≫ epicatechin ⩾ catechin > gallocatechin ⩾ epigallocatechin. All free energy changes possessed negative sign indicating the spontaneity of catechin-HSA systems formation. The binding distances between the donor (HSA) and the acceptors (catechins) estimated by the Förster theory revealed that non-radiation energy transfer from HSA to catechins occurred with high possibility. According to results obtained by native PAGE, the galloylated catechins increased the electrophoretic mobility of HSA, which indicated the change in the molecular charge of HSA, whilst the non-galloylated catechins caused no changes. The ability of aggregation and cross-linking of tested catechins with HSA was not proved by SDS-PAGE. The relationship between the structure characteristics of all tested catechins (e.g. presence of the galloyl moiety on the C-ring, the number of hydroxyl groups on the B-ring, and the spatial arrangement of the substituents on the C-ring) and their binding properties to HSA is discussed. The presented study contributes to the current knowledge in the area of protein-ligand binding, particularly catechin-HSA interactions.

  14. Exploring the interactions of decabrominateddiphenyl ether and tetrabromobisphenol A with human serum albumin.

    PubMed

    Wang, Yan-Qing; Zhang, Hong-Mei; Cao, Jian

    2014-09-01

    Decabrominateddiphenyl ether (deca-BDE) and tetrabromobisphenol A (TBBPA) are known as brominated flame-retardants, which are commonly found in the environment. The binding mechanisms of deca-BDE and TBBPA with human serum albumin (HSA) are still unknown. In this report, the interactions of deca-BDE and TBBPA with HSA were investigated using different spectroscopic methods and molecular modeling. The experimental results indicated the formation of complexes between deca-BDE/TBBPA and HSA with different affinity. These interactions affected the secondary structure of HSA. Thermodynamic investigations revealed that hydrophobic forces mainly drove the binding interactions of deca-BDE/TBBPA with HSA. For TBBPA, hydrogen-bonding interactions were also involved in the binding process of TBBPA with HSA. According to the analysis of experimental and theoretical data, we concluded that the binding site of deca-BDE to HSA located in the subdomain IB, while TBBPA was near to subdomain IIA and Trp-214. The binding interactions of deca-BDE and TBBPA with the most prominent carrier protein in the human circulatory system could influence mechanisms of their biochemical processes. Thus, these binding interactions can play central roles in studying the distribution and toxicity mechanisms of brominated flame-retardants. PMID:25194328

  15. The effect of Berberine on the secondary structure of human serum albumin

    NASA Astrophysics Data System (ADS)

    Li, Ying; He, WenYing; Tian, Jianniao; Tang, Jianghong; Hu, Zhide; Chen, Xingguo

    2005-05-01

    The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the presence of Berberine. The Scatchard's plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one binding site with the binding constant is 4.071(±0.128)×10 4, 3.741(±0.089)×10 4, 3.454(±0.110)×10 4 M -1, respectively. The protein conformation is altered (FT-IR and CD data) with reductions of α-helices from 54 to 47% for free HSA to 45-32% and with increases of turn structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain IIA, that is, high affinity site (site II).

  16. Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

    2008-11-01

    Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

  17. Anesthetic 2,2,2-trifluoroethanol induces amyloidogenesis and cytotoxicity in human serum albumin.

    PubMed

    Naeem, Aabgeena; Iram, Afshin; Bhat, Sheraz Ahmed

    2015-08-01

    Trifluoroethanol (TFE) mimics the membrane environments as it simulates the hydrophobic environment and better stabilizes the secondary structures in peptides owing to its hydrophobicity and hydrogen bond-forming properties. Its dielectric constant approximates that of the interior of proteins and is one-third of that of water. Human serum albumin (HSA) is a biological transporter. The effect of TFE on HSA gives the clue about the conformational changes taking place in HSA on transport of ligands across the biological membranes. At 25% (v/v) and 60% (v/v) TFE, HSA exhibits non-native β-sheet, altered tryptophan fluorescence, exposed hydrophobic clusters, increased thioflavin T fluorescence and prominent red shifted Congo red absorbance, and large hydrodynamic radii suggesting the aggregate formation. Isothermal titration calorimetric results indicate weak binding of TFE and HSA. This suggests that solvent-mediated effects dominate the interaction of TFE and HSA. TEM confirmed prefibrillar at 25% (v/v) and fibrillar aggregates at 60% (v/v) TFE. Comet assay of prefibrillar aggregates showed DNA damage causing cell necrosis hence confirming cytotoxic nature. On increasing concentration of TFE to 80% (v/v), HSA showed retention of native-like secondary structure, increased Trp and ANS fluorescence, a transition from β-sheet to α-helix. Thus, TFE at high concentration possess anti- aggregating potency.

  18. Exploring the binding mechanism of ondansetron hydrochloride to serum albumins: spectroscopic approach.

    PubMed

    B, Sandhya; Hegde, Ashwini H; K C, Ramesh; J, Seetharamappa

    2012-02-01

    The mechanism of interaction of ondansetron hydrochloride (OND) to serum albumins [bovine serum albumin (BSA) and human serum albumin (HSA)] was studied for the first time employing fluorimetric, circular dichroism, FTIR and UV-vis absorption techniques under the simulated physiological conditions. Fluorimetric results were utilized to investigate the binding and conformational characteristics of protein upon interaction with varying concentrations of the drug. Higher binding constant values revealed the strong interaction between the drug and protein while the number of binding sites close to unity indicated single class of binding site for OND in protein. Thermodynamic results revealed that both hydrogen bond and hydrophobic interactions played a major role in stabilizing drug-protein complex. Site marker competitive experiments indicated that the OND bound to albumins at subdomin II A (Sudlow's site I). Further, the binding distance between OND and serum albumin was calculated based on the Förster's theory of non-radioactive energy transfer and found to be 2.30 and 3.41 nm, respectively for OND-BSA and OND-HSA. The circular dichroism data revealed that the presence of OND decreased the α-helix content of serum albumins. 3D-fluorescence results also indicated the conformational changes in protein upon interaction with OND. Further, the effects of some cations have been investigated in the interaction of drug to protein.

  19. Structural basis of transport of lysophospholipids by human serum albumin

    SciTech Connect

    Guo, Shihui; Shi, Xiaoli; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Bian, Chuanbing; Huang, Mingdong

    2010-10-08

    Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a K{sub d} (dissociation constant) of 5.6 {micro}M. The presence of FA (myristate) decreases this binding affinity (K{sub d} of 12.9 {micro}M). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

  20. Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation.

    PubMed

    Li, M; Hagerman, A E

    2015-01-01

    (-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10-100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.

  1. Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

    PubMed

    Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

    2014-03-01

    Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

  2. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin

    NASA Astrophysics Data System (ADS)

    Jupin, M.; Michiels, P. J.; Girard, F. C.; Spraul, M.; Wijmenga, S. S.

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  3. 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

  4. Orientation of the water molecules of hydration of human serum albumin.

    PubMed

    van Oss, C J; Good, R J

    1988-04-01

    Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is approximately 98% in the first layer of hydration and approximately 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.

  5. The Role of Protein Excipient in Driving Antibody Responses to Erythropoietin.

    PubMed

    Christie, Merry; Peritt, David; Torres, Raul M; Randolph, Theodore W; Carpenter, John F

    2015-12-01

    Human serum albumin (HSA) is an excipient present in formulations of several recombinant protein products that are approved for clinical use. We investigated the relative contributions of HSA and HSA particles to the generation of antibody responses against recombinant human erythropoietin (rhEPO) and the excipient HSA itself. Protein samples were characterized before injection for quantities of monomeric proteins, soluble protein aggregates, and nano- and micron-sized particles. rhEPO, containing various concentrations of HSA particles, were injected three times a week for 8 weeks into mice. Hematocrits and the production of anti-rhEPO and anti-HSA antibodies were determined at various time points. Levels of antibodies against rhEPO in mice injected with HSA-containing rhEPO were higher than those in mice treated with HSA-free rhEPO. Mice injected with formulations that contained particles of HSA produced strong anti-HSA antibody responses; whereas these responses were greatly reduced when particle-free formulations were administered. In contrast, anti-rhEPO antibody responses were not affected by the presence of particles.

  6. Maximizing the relaxivity of Gd-complex by synergistic effect of HSA and carboxylfullerene.

    PubMed

    Zhen, Mingming; Zheng, Junpeng; Ye, Lei; Li, Shumu; Jin, Chan; Li, Kai; Qiu, Dong; Han, Hongbin; Shu, Chunying; Yang, Yongji; Wang, Chunru

    2012-07-25

    Macromolecular magnetic resonance imaging (MRI) contrast agent Gd-DTPA-HSA (DTPA, diethylene triamine pentacetate acid; HSA, human serum albumin) as a model has been successfully conjugated with trimalonic acid modified C60 for contrast enhancement at clinically used magnetic field strength. The Gd-DTPA-HSA-C60 conjugate exhibit maximal relaxivity (r1 = 86 mM(-1) s(-1) at 0.5 T, 300 K) reported so far, which is much superior to that of the control Gd-DTPA-HSA (r1 = 38 mM(-1 )s(-1)) under the same condition and comparable to the theoretical maximum (r1 = 80-120 mM(-1) s(-1), at 20 MHz and 298 K), indicating the synergistic effect of HSA and carboxylfullerene on the increased contrast enhancement. TEM characterization reveals that both Gd-DTPA-HSA-C60 and Gd-DTPA-HSA can penetrate the cells via endocytosis and trans-membrane, respectively, suggesting the potential to sensitively image the events at the cellular and subcellular levels. In addition, the fusion of fullerene with Gd-DTPA-HSA will further endow the resulting complex with photodynamic therapy (PDT) property and thus combine the modalities of therapy (PDT) and diagnostic imaging (MRI) into one entity. More importantly, the payloaded Gd-DTPA may substitute for other more stable Gd-DOTA and HSA as a theranostic package can further work as a drug delivery carrier and effectively control drug release through proteolysis.

  7. Human plasma lipocalins and serum albumin: Plasma alternative carriers?

    PubMed

    di Masi, Alessandra; Trezza, Viviana; Leboffe, Loris; Ascenzi, Paolo

    2016-04-28

    Lipocalins are an evolutionarily conserved family of proteins that bind and transport a variety of exogenous and endogenous ligands. Lipocalins share a conserved eight anti-parallel β-sheet structure. Among the different lipocalins identified in humans, α-1-acid glycoprotein (AGP), apolipoprotein D (apoD), apolipoprotein M (apoM), α1-microglobulin (α1-m) and retinol-binding protein (RBP) are plasma proteins. In particular, AGP is the most important transporter for basic and neutral drugs, apoD, apoM, and RBP mainly bind endogenous molecules such as progesterone, pregnenolone, bilirubin, sphingosine-1-phosphate, and retinol, while α1-m binds the heme. Human serum albumin (HSA) is a monomeric all-α protein that binds endogenous and exogenous molecules like fatty acids, heme, and acidic drugs. Changes in the plasmatic levels of lipocalins and HSA are responsible for the onset of pathological conditions associated with an altered drug transport and delivery. This, however, does not necessary result in potential adverse effects in patients because many drugs can bind both HSA and lipocalins, and therefore mutual compensatory binding mechanisms can be hypothesized. Here, molecular and clinical aspects of ligand transport by plasma lipocalins and HSA are reviewed, with special attention to their role as alterative carriers in health and disease. PMID:26951925

  8. Structure and properties of licochalcone A-human serum albumin complexes in solution: a spectroscopic, photophysical and computational approach to understand drug-protein interaction.

    PubMed

    Monti, Sandra; Manet, Ilse; Manoli, Francesco; Marconi, Giancarlo

    2008-11-28

    In the present contribution we address the study of the interaction of a flavonoid-derivative licochalcone A (LA) with human serum albumin (HSA). The application of circular dichroism, UV-Vis absorption, fluorescence and laser flash photolysis combined with molecular mechanics, molecular dynamics and quantum mechanical calculations of rotational strength afforded a clear picture of the modes of association of the LA neutral molecule to HSA, evidencing specific interactions with protein amino acids and their photophysical consequences. The drug is primarily associated in subdomain IIA where a strong interaction with Trp214 is established. At least two different positions of LA with respect to tryptophan are possible, one with the phenolic ring of the drug facing the aromatic ring of Trp214 and the other with the methoxyphenolic ring of LA in proximity to Trp214. In both cases LA is at ca. 4 angstroms from Trp214. This vicinity does not affect much the S1 singlet state deactivation of the bound drug, which exhibits a slightly higher fluorescence quantum yield and fluorescence lifetime on the order of that of the free molecule. The LA triplet lifetime appears to be somewhat shortened in this site. The secondary binding site is in subdomain IIIA. Here, the carbonyl group of LA experiences a strong H-bond with the OH-phenolic substituent of Tyr411. This interaction reduces substantially the LA molecular degrees of freedom, thereby determining a decrease of both radiative and nonradiative rate constants for decay of the singlet. The overall rigidity of the structure causes a lengthening of the triplet lifetime. PMID:18989470

  9. Prion like behavior of HSA-hydroxylated MWCNT interface.

    PubMed

    Sekar, Gajalakshmi; Sivakumar, A; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2016-08-01

    Carbon nanotubes (CNTs) with unique and outstanding properties were expected to revolutionize various aspects of the biomedical sector. Interaction studies of proteins with functionalized CNTs would shed light on their toxicological aspects upon entering the human body. Hyperchromicity of the UV-Visible spectra and declining fluorescence potential of HSA on interaction with CNTs suggested ground state complex to exist between them. Synchronous and 3D spectral features of CNT-HSA system proposed their possible binding site to occur nearby Trp and Tyr residues. FTIR and FT-Raman spectra showed a shift in the amide band region that proportionate the possible alteration to occur in the alpha-helical structures. CD far and near spectra showed loss of alpha-helical structures and shift in the Trp position of the polypeptide backbone. CNT's UV and FTIR band showed shift on interaction with HSA, which conveys the possible aggregation of CNTs in the presence of protein. The promoting role of CNTs against HSA fibril formation has been confirmed by spectroscopic and microscopic evaluations. Secondary conformational changes, besides the existence of increased beta-sheet structures of HSA amyloid fibrils, remain similar to the amyloid behavior of Prion protein. Hence, HSA fibril-CNT interface predominates the possible mechanism for several amyloid-related disorders concerning their toxic accumulations in the body. PMID:27314539

  10. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

    PubMed Central

    Haberland, M E; Fogelman, A M

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor. PMID:3857610

  11. Interactions Between Sirolimus and Anti-Inflammatory Drugs: Competitive Binding for Human Serum Albumin

    PubMed Central

    Khodaei, Arash; Bolandnazar, Soheila; Valizadeh, Hadi; Hasani, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: The aim of the present study was investigating the effects of three anti-inflammatory drugs, on Sirolimus protein biding. The binding site of Sirolimus on human serum albumin (HSA) was also determined. Methods: Six different concentrations of Sirolimus were separately exposed to HSA at pH 7.4 and 37°C. Ultrafiltration method was used for separating free drug; then free drug concentrations were measured by HPLC. Finally, Sirolimus protein binding parameters was calculated using Scatchard plots. The same processes were conducted in the presence of NSAIDs at lower concentration of albumin and different pH conditions. To characterize the binding site of Sirolimus on albumin, the free concentration of warfarin sodium and Diazepam, site I and II specific probes, bound to albumin were measured upon the addition of increasing Sirolimus concentrations. Results: Based on the obtained results presence of Diclofenac, Piroxicam and Naproxen, could significantly decrease the percentage of Sirolimus protein binding. The Binding reduction was the most in the presence of Piroxicam. Sirolimus-NSAIDs interactions were increased in higher pH values and also in lower albumin concentrations. Probe displacement study showed that Sirolimus may mainly bind to site I on albumin molecule. Conclusion: More considerations in co-administration of NSAIDs and Sirolimus is recommended. PMID:27478785

  12. Significance of Total Protein, Albumin, Globulin, Serum Effusion Albumin Gradient and LDH in the Differential Diagnosis of Pleural Effusion Secondary to Tuberculosis and Cancer

    PubMed Central

    Sharma, Ashish; Das, Biswajit; Mallick, Ayaz K; Kumar, Amit

    2016-01-01

    Introduction Lung cancer and Pulmonary tuberculosis are two major public health problems associated with significant morbidity and mortality worldwide particularly in low and middle income countries like India. Wrong diagnosis of lung cancer cases as pulmonary tuberculosis in primary health care system delays the onset of anti-cancer chemotherapy as well as initiation of DOTS thus increasing complication and mortality rate in malignancy patients. In this context easy, cost effective diagnostic tool at primary level must be the priority and need of hour. Aim To study and evaluate any significance of biochemical parameters (total protein, albumin, globulin, serum effusion albumin gradient, LDH) in serum and pleural effusion secondary to tuberculosis and lung cancer. Materials and Methods A case control study was carried out on patients attending OPD and IPD, Department of Pulmonary Medicine, RMCH. Hundred cases of Tuberculosis effusion, 50 cases of Malignant effusion and 100 age and sex matched apparently healthy controls were taken for correlation of biochemical parameters (total protein, albumin, globulin, serum effusion albumin gradient, LDH) and statistically evaluated to find any significance between tuberculosis, lung cancer and control group. Blood and pleural fluid samples were collected and then subjected to assessment of parameters (total protein, albumin, LDH) by using EM360 Autoanalyser and kits were supplied by Transasia diagnostics. Globulin and Serum Effusion Albumin Gradient (SEAG) was calculated mathematically. Statistical Analysis Data is presented as mean ± SD. Comparison of serum and pleural fluid levels (of taken parameters) were done in TB, Lung Cancer and Control groups by ANOVA and students t-test. The p-value <0.05 were considered as statistically significant. Results We found serum-total protein, albumin, globulin to be significantly higher in TB group than lung cancer group but serum LDH was higher in lung cancer group (in all parameters p

  13. Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

    PubMed

    Tayyab, Saad; Izzudin, Mohamad Mirza; Kabir, Md Zahirul; Feroz, Shevin R; Tee, Wei-Ven; Mohamad, Saharuddin B; Alias, Zazali

    2016-09-01

    Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlow's site I, located in subdomain IIA of HSA, as the preferred binding site of AXT.

  14. Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

    PubMed

    Tayyab, Saad; Izzudin, Mohamad Mirza; Kabir, Md Zahirul; Feroz, Shevin R; Tee, Wei-Ven; Mohamad, Saharuddin B; Alias, Zazali

    2016-09-01

    Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlow's site I, located in subdomain IIA of HSA, as the preferred binding site of AXT. PMID:27424099

  15. Acute metabolic acidosis decreases muscle protein synthesis but not albumin synthesis in humans.

    PubMed

    Kleger, G R; Turgay, M; Imoberdorf, R; McNurlan, M A; Garlick, P J; Ballmer, P E

    2001-12-01

    Chronic metabolic acidosis induces negative nitrogen balance by either increased protein breakdown or decreased protein synthesis. Few data exist regarding effects of acute metabolic acidosis on protein synthesis. We investigated fractional synthesis rates (FSRs) of muscle protein and albumin, plasma concentrations of insulin-like growth factor-I (IGF-I), thyroid-stimulating hormone (TSH), and thyroid hormones (free thyroxin [fT(4)] and triiodothyronine [fT(3)]) in seven healthy human volunteers after a stable controlled metabolic period of 5 days and again 48 hours later after inducing metabolic acidosis by oral ammonium chloride intake (4.2 mmol/kg/d divided in six daily doses). Muscle and albumin FSRs were obtained by the [(2)H(5)ring]phenylalanine flooding technique. Ammonium chloride induced a significant decrease in pH (7.43 +/- 0.02 versus 7.32 +/- 0.04; P < 0.0001) and bicarbonate concentration (24.6 +/- 1.6 versus 16.0 +/- 2.7 mmol/L; P < 0.0001) within 48 hours. Nitrogen balance decreased significantly on the second day of acidosis. The FSR of muscle protein decreased (1.94 +/- 0.25 versus 1.30 +/- 0.39; P < 0.02), whereas the FSR of albumin remained constant. TSH levels increased significantly (1.1 +/- 0.5 versus 1.9 +/- 1.1 mU/L; P = 0.03), whereas IGF-I, fT(4), and fT(3) levels showed no significant change. We conclude that acute metabolic acidosis for 48 hours in humans induces a decrease in muscle protein synthesis, which contributes substantially to a negative nitrogen balance. In contrast to prolonged metabolic acidosis of 7 days, a short period of acidosis in the present study did not downregulate albumin synthesis.

  16. Gold Nanoparticle-Based Facile Detection of Human Serum Albumin and Its Application as an INHIBIT Logic Gate.

    PubMed

    Huang, Zhenzhen; Wang, Haonan; Yang, Wensheng

    2015-05-01

    In this work, a facile colorimetric method is developed for quantitative detection of human serum albumin (HSA) based on the antiaggregation effect of gold nanoparticles (Au NPs) in the presence of HSA. The citrate-capped Au NPs undergo a color change from red to blue when melamine is added as a cross-linker to induce the aggregation of the NPs. Such an aggregation is efficiently suppressed upon the adsorption of HSA on the particle surface. This method provides the advantages of simplicity and cost-efficiency for quantitative detection of HSA with a detection limit of ∼1.4 nM by monitoring the colorimetric changes of the Au NPs with UV-vis spectroscopy. In addition, this approach shows good selectivity for HSA over various amino acids, peptides, and proteins and is qualified for detection of HSA in a biological sample. Such an antiaggregation effect can be further extended to fabricate an INHIBIT logic gate by using HSA and melamine as inputs and the color changes of Au NPs as outputs, which may have application potentials in point-of-care medical diagnosis. PMID:25850684

  17. Spectroscopic investigation of the interaction between human serum albumin and three organic acids

    NASA Astrophysics Data System (ADS)

    Liu, Yuan; Xie, Meng-Xia; Jiang, Min; Wang, Ying-Dian

    2005-07-01

    The interactions of human serum albumin (HSA) with sinapic acid (SA), gallic acid (GA) and shikimic acid (SI) were investigated by fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of GA at the molar ratio of drug to HSA ranging from 0.1 to 30, and their binding constant ( KA) is 1.1 × 10 4 M -1. While one HSA molecule combined with one or two molecule of SA at the molar ratio of drug to HSA ranging from 0.1 to 4.26 or 4.26 to 30, and their binding affinities ( KA) are 1.92 × 10 3 M -1 and 6.87 × 10 8 M -1, respectively. There is no specific interaction between HSA and SI. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structures induced by drugs were estimated. The drug-protein combination brought gradual reductions of the protein α-helix structure with increasing the concentrations of SA and GA, but SI did not change the protein secondary structure. From the fluorescence and FT-IR results, the binding mode was discussed in relation to the structures of the organic acids.

  18. Adsorption of HSA, IgG and laminin-1 on model titania surfaces--effects of glow discharge treatment on competitively adsorbed film composition.

    PubMed

    Santos, Olga; Svendsen, Ida E; Lindh, Liselott; Arnebrant, Thomas

    2011-10-01

    This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA protein mixture. Film elutability showed that 30 min of SDS interaction resulted in almost complete removal of adsorbed films. No difference in the total adsorbed amounts of individual proteins, or from the mixture, was observed between untreated and glow discharge treated titania surfaces. However, the composition of the adsorbed films from the mixture differed between the untreated and glow discharge treated substrata. On glow discharge-treated titania the fraction of HSA increased, the fraction of laminin-1 decreased and the fraction of IgG was unchanged compared to the adsorption on the untreated titania, which was attributed to protein-protein interactions and competitive/associative adsorption behaviour.

  19. Isolation and characterization of serum albumin from Camelus dromedarius

    PubMed Central

    MALIK, AJAMALUDDIN; AL-SENAIDY, ABDULRAHMAN; SKRZYPCZAK-JANKUN, EWA; JANKUN, JERZY

    2013-01-01

    Serum albumin constitutes 35–50 mg/ml of plasma proteins and performs various physiological activities including the regulation of osmotic pressure on blood, maintaining buffering of the blood pH, carrying different fatty acids and other small molecules, such as bilirubin, hormones, drugs and metal ions, as well as participating in immunological responses. Serum albumin is an extensively used protein in biotechnological and pharmaceutical industries. The camel (Camelus dromedarius) is well tailored to successfully survive in extremely hot and dry climates. Plasma osmolality in the camel increases during water-deprived conditions. In such circumstances serum albumin is crucial in the regulation of blood pressure. The study of biochemical, biophysical and immunological aspects of camel serum albumin (CSA) are likely to provide molecular insights into camel physiology and may render it an alternative to human serum albumin (HSA) and bovine serum albumin (BSA) in all cases. However, these proteins are currently not available or cannot be utilized due to a variety of considerations. In this study, 12 mg of highly pure CSA was obtained from 1 ml plasma. Coomassie Brilliant Blue staining of SDS-PAGE yielded one band and RP-HPLC results revealed a single sharp peak, indicating homogenous preparation of the CSA. The charge/mass ratio and surface hydrophobicity of the CSA was similar to that of BSA. Mass spectrometry analysis of the purified protein confirmed the identity of CSA. PMID:24137219

  20. Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

    PubMed

    Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

    2016-09-01

    Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively. PMID:27122488

  1. Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

    PubMed

    Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

    2016-09-01

    Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.

  2. Biophysical studies of interaction between hydrolysable tannins isolated from Oenothera gigas and Geranium sanguineum with human serum albumin.

    PubMed

    Sekowski, Szymon; Ionov, Maksim; Kaszuba, Mateusz; Mavlyanov, Saidmukhtar; Bryszewska, Maria; Zamaraeva, Maria

    2014-11-01

    Tannins, secondary plant metabolites, possess diverse biological activities and can interact with biopolymers such as lipids or proteins. Interactions between tannins and proteins depend on the structures of both and can result in changes in protein structure and activity. Because human serum albumin is the most abundant protein in plasma and responsible for interactions with important biological compounds (e.g. bilirubin) and proper blood pressure, therefore, it is very important to investigate reactions between HSA and tannins. This paper describes the interaction between human serum albumin (HSA) and two tannins: bihexahydroxydiphenoyl-trigalloylglucose (BDTG) and 1-O-galloyl-4,6-hexahydroxydiphenoyl-β-d-glucose (OGβDG), isolated from Geranium sanguineum and Oenothera gigas leafs, respectively. Optical (spectrofluorimetric) and chiral optical (circular dichroism) methods were used in this study. Fluorescence analysis demonstrated that OGβDG quenched HSA fluorescence more strongly than BDTG. Both OGβDG and BDTG formed complexes with albumin and caused a red shift of the fluorescence spectra but did not significantly change the protein secondary structure. Our studies clearly demonstrate that the tested tannins interact very strongly with human serum albumin (quenching constant K=88,277.26±407.04 M(-1) and K=55,552.67±583.07 M(-1) respectively for OGβDG and BDTG) in a manner depending on their chemical structure.

  3. Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.).

    PubMed

    Smedsrud, T; Dannevig, B H; Tolleshaug, H; Berg, T

    1984-01-01

    The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal. PMID:6500136

  4. Study of the interaction of C60 fullerene with human serum albumin in aqueous solution

    SciTech Connect

    Li, Song; Zhao, Xiongce; Mo, Yiming; Cummings, Peter T; Heller, William T

    2013-01-01

    Concern about the toxicity of engineered nanoparticles, such as the prototypical nanomaterial C60 fullerene, continues to grow. While evidence continues to mount that C60 and its derivatives may pose health hazards, the specific molecular interactions of these particles with biological macromolecules require further investigation. To better understand the interaction of C60 with proteins, the protein human serum albumin (HSA) was studied in solution with C60 at C60:HSA molar ratios ranging from 1:2 to 4:1. HSA is the major protein component of blood plasma and plays a role in a variety of functions, such as the maintenance of blood pH and pressure. The C60-HSA interaction was probed by a combination of circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and atomistic molecular dynamics (MD) simulations to understand C60-driven changes in the structure of HSA in solution. The CD spectroscopy demonstrates that the secondary structure of the protein decreases in -helical content in response to the presence of C60. Similarly, C60 produces subtle changes in the solution conformation of HSA, as evidenced by the SANS data and MD. The data do not indicate that C60 is causing a change in the oligomerization state of the protein. Taken together results demonstrate that C60 interacts with HSA, but it does not strongly perturb the structure of the protein by unfolding it or inducing aggregation, suggesting a mechanism for transporting C60 throughout the body to accumulate in various tissues.

  5. Potential toxicity of sulfanilamide antibiotic: binding of sulfamethazine to human serum albumin.

    PubMed

    Chen, Jiabin; Zhou, Xuefei; Zhang, Yalei; Gao, Haiping

    2012-08-15

    Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. The interaction between human serum albumin (HSA) and sulfamethazine (SMZ) was investigated by capillary electrophoresis, fluorescence spectrometry, and circular dichroism. The binding constant and site were determined to be 1.09×10(4) M(-1) and 1.14 at 309.5 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change, where the electrostatic interaction and hydrogen bond were the dominant binding force. The binding distance between SMZ and tryptophan residue of HSA was obtained to be 3.07 nm according to Fǒrster non-radioactive energy transfer theory. The site marker competition revealed that SMZ bound into subdomain IIA of HSA. The binding of SMZ induced the unfolding of the polypeptides of HSA and transferred the secondary conformation of HSA. The equilibrium dialysis showed that only 0.13 mM SMZ decreased vitamin B(2) by 38% transported on the HSA. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage. PMID:22750172

  6. A Comprehensive Computational Study of the Interaction between Human Serum Albumin and Fullerenes.

    PubMed

    Leonis, Georgios; Avramopoulos, Aggelos; Papavasileiou, Konstantinos D; Reis, Heribert; Steinbrecher, Thomas; Papadopoulos, Manthos G

    2015-12-01

    Human serum albumin (HSA) is the most abundant blood plasma protein, which transports fatty acids, hormones, and drugs. We consider nanoparticle-HSA interactions by investigating the binding of HSA with three fullerene analogs. Long MD simulations, quantum mechanical (fragment molecular orbital, energy decomposition analysis, atoms-in-molecules), and free energy methods elucidated the binding mechanism in these complexes. Such a systematic study is valuable due to the lack of comprehensive theoretical approaches to date. The main elements of the mechanism include the following: binding to IIA site results in allosteric modulation of the IIIA and heme binding sites with an increase in α-helical structure of IIIA. Fullerenes displayed high binding affinities for HSA; therefore, HSA can be used as a fullerene carrier, facilitating any toxic function the fullerene may exert. Complex formation is driven by hydrogen bonding, van der Waals, nonpolar, charge transfer, and dispersion energy contributions. Proper functionalization of C60 has enhanced its binding to HSA by more than an order of magnitude. This feature may be important for biological applications (e.g., photodynamic therapy of cancer). Satisfactory agreement with relevant experimental and theoretical data has been obtained.

  7. Study the interactions between human serum albumin and two antifungal drugs: fluconazole and its analogue DTP.

    PubMed

    Zhang, Shao-Lin; Yao, Huankai; Wang, Chenyin; Tam, Kin Y

    2014-11-01

    Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern-Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10(4)L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug-protein. The effect of metal ions on the binding constants of fluconazole-HSA complex suggested that the presence of Mg(2+) and Zn(2+) ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.

  8. Methyl-triclosan binding to human serum albumin: multi-spectroscopic study and visualized molecular simulation.

    PubMed

    Lv, Wenjuan; Chen, Yonglei; Li, Dayong; Chen, Xingguo; Leszczynski, Jerzy

    2013-10-01

    Methyl-triclosan (MTCS), a transformation product and metabolite of triclosan, has been widely spread in environment through the daily use of triclosan which is a commonly used anti-bacterial and anti-fungal substance in consumer products. Once entering human body, MTCS could affect the conformation of human serum albumin (HSA) by forming MTCS-HSA complex and alter function of protein and endocrine in human body. To evaluate the potential toxicity of MTCS, the binding mechanism of HSA with MTCS was investigated by UV-vis absorption, circular dichroism and Fourier transform infrared spectroscopy. Binding constants, thermodynamic parameters, the binding forces and the specific binding site were studied in detail. Binding constant at room tempreture (T = 298K) is 6.32 × 10(3)L mol(-1); ΔH(0), ΔS(0) and ΔG(0) were 22.48 kJ mol(-1), 148.16 J mol(-1)K(-1) and -21.68 kJ mol(-1), respectively. The results showed that the interactions between MTCS and HSA are mainly hydrophobic forces. The effects of MTCS on HSA conformation were also discussed. The binding distance (r = 1.2 nm) for MTCS-HSA system was calculated by the efficiency of fluorescence resonance energy transfer. The visualized binding details were also exhibited by molecular modeling method and the results could agree well with that from the experimental study.

  9. Inhibitory Effects of Some Carbohydrates on Nano-Globular Aggregation of both Normal and Glycated Albumin

    PubMed Central

    Moosavi-Movahedi, Ali Akbar; Sattarahmady, Naghmeh; Sharifi, Esmaeil; Heli, Hossein

    2016-01-01

    Background: Protein aggregation is one of the important, common and troubling problems in biotechnology, pharmaceutical industries and amyloid-re-lated disorders. Methods: In the present study, the inhibitory effects of some carbohydrates (alginate, β-cyclodextrin and trehalose) on the formation of nano-globular aggregates from normal (HSA) and glycated (GHSA) human serum albumin were studied; when the formation of aggregates was induced by the simultaneous heating and addition of dithiotheritol. For the investigations, the biophysical methods of UV-vis spectrophotometry, circular dichroism spectroscopy, transmission electron microscopy and tensiometry were employed. Results: The effect of inhibitory mechanism of these inhibitors on the aggregation of HSA and GHSA was expressed and compared together. Conclusion: The results showed that the nucleus formation step of the aggregation process of HSA and GHSA was different in the presence of alginate (compared to β-cyclodextrin and trehalose). The inhibition efficiencies of the carbohydrates on the aggregate formation of HSA and GHSA were different, arising from the differences in the hydrophobicities of HSA and GHSA, and also, the differences between HSA- and GHSA-carbohydrate interactions. PMID:27563425

  10. Spectroscopic characterization of the binding mechanism of fluorescein and carboxyfluorescein in human serum albumin

    NASA Astrophysics Data System (ADS)

    Sulaiman, Saba A. J.; Kulathunga, H. Udani; Abou-Zied, Osama K.

    2015-03-01

    Fluorescein (FL) and some of its precursors have proven to be effective fluorescent tracers in pharmaceutical and medical applications owing to their high quantum yield of fluorescence in physiological conditions and their high membrane permeability. In order to protect FL from metabolic effects during the process of its delivery, human serum albumin (HSA) has been used as a carrier because of its compatibility with the human body. In the present work, we used spectroscopic methods to characterize the binding mechanisms of FL and one of its derivatives, 5(6)- carboxyfluorescein (CFL), in the HSA protein. The absorbance change of the two ligands (FL and CFL) was quantified as a function of the HSA concentration and the results indicate a moderate binding strength for the two ligands inside HSA (1.00 +/- 0.12 x 104 M-1). The quenching effect of FL(CFL) on the fluorescence intensity of W214 (the sole tryptophan in HSA) indicates that FL and CFL occupy Site I in the protein which is known to bind several hydrophobic drugs. By performing site-competitive experiments, the location of the ligands is determined to be similar to that of the anticoagulant drug warfarin. At higher ratios of [ligand]/[HSA], we observed an upward curvature in the Stern-Volmer plots which indicates that the ligands occupy more pockets in Site I, close to W214. Our results indicate that both ligands bind in HSA with a moderate strength that should not affect their release when used as fluorescent reporters. The chemical and physical identities of the two ligands are also preserved inside the HSA binding sites.

  11. Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

    PubMed Central

    Frahm, Grant E.; Smith, Daryl G. S.; Kane, Anita; Lorbetskie, Barry; Cyr, Terry D.; Girard, Michel; Johnston, Michael J. W.

    2014-01-01

    The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well

  12. Interference-free determination of ischemia-modified albumin using quantum dot coupled X-ray fluorescence spectroscopy.

    PubMed

    Luo, Yang; Wang, Chaoming; Jiang, Tianlun; Zhang, Bo; Huang, Junfu; Liao, Pu; Fu, Weiling

    2014-01-15

    Ischemia-modified protein (IMA) is the most sensitive diagnostic biomarker of ischemic heart disease, but differentiation of IMA from human serum albumin (HSA), a ubiquitous serum protein, is still challenging owing to the shared antigenicity. In this investigation, we developed a rapid and interference-free approach for IMA determination using quantum dots-coupled X-ray Fluorescence Spectroscopy (Q-XRF). In a typical Q-XRF assay, serum total HSA is quantified using quantum dot-coupled sandwich immunoassay, and intact HSA (iHSA) is determined using a XRF spectroscopy, by measuring XRF intensity of Co (II) bonded to iHSA. IMA concentration is automatically determined within 30 min by calculating the difference between total HSA and iHSA. This strategy can effectively eliminate the interference from native HSA level. Results show that no significant influences have been observed from hemolysis or high levels of cholesterol (7 mg/L), triglyceride (5.2 mg/L), IgG (10 g/L), and fibrinogen (4 g/L). A linearity of 1-100mg/mL is obtained in iHSA determination using XRF (r(2)=0.979). The proposed Q-XRF assay demonstrates a lowest detection limit of 0.05 U/mL. Receiver-operating characteristic (ROC) curves reveal that Q-XRF assay provide an improved sensitivity than ACB assay (95.9% vs. 82.9%) in differentiating ischemic patients from health individuals, at an optimal cutoff point of 79.2U/mL. The proposed approach provides a new strategy for interference-free, simple and rapid evaluation of IMA concentration by combining sandwich immunoassay and XRF spectroscopy.

  13. O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin.

    PubMed

    Nakagawa, Akito; Komatsu, Teruyuki; Huang, Yubin; Lu, Gang; Tsuchida, Eishun

    2007-01-01

    Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.

  14. Interaction of drugs with bovine and human serum albumin

    NASA Astrophysics Data System (ADS)

    Sułkowska, Anna

    2002-09-01

    The study on the interaction of antithyroid drugs: 2-mercapto-1-methylimidazole (Methimazole, MMI) and 6 n-propyl-2-thiouracil (PTU) with two kinds of serum albumin: bovine (BSA) and human (HSA) has been undertaken. Fluorescence emission spectra of serum albumin in the presence of MMI or PTU, recorded at the excitation wavelengths 280 and 295 nm, clearly show that the studied drugs act as quenchers. A decrease in fluorescence intensity at 340 or 350 nm, when excited at 280 or 295 nm, respectively, is attributed to changes in the environment of the protein fluorophores caused to the presence of the ligand. The 295 nm lights excites tryptophan residues, while the 280 nm lights excites both tryptophan and tyrosine residues. A comparison of quenching effects, when protein is excited at 295 and 280 nm, reveals that the tryptophanyl group interacts with the ligand. The differences in interactions of pyrimidine derivatives with HSA and BSA were observed using spectrofluorimetry technique. As the HSA structure contains only one tryptophanyl residue (Trp 214), while BSA has two ones (Trp 135 and Trp 214), the similar decrease of fluorescence points at the subdomain IIA, where Trp 214 was located, as a binding site of the studied drugs.

  15. Human serum albumin-coated gold nanoparticles for selective extraction of lysozyme from real-world samples prior to capillary electrophoresis.

    PubMed

    Yeh, Pei-Rong; Tseng, Wei-Lung

    2012-12-14

    This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complex matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limit of detection at a signal-to-noise ratio of 3 for Lyz was down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of Lyz in hen egg white, human milk, and human tear. Also, this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  16. Fluorescence spectroscopic study of serum albumin-bromadiolone interaction: fluorimetric determination of bromadiolone.

    PubMed

    Deepa, Subbiah; Mishra, Ashok K

    2005-07-01

    Bromadiolone (BRD), a substituted 4-hydroxycoumarin derivative, is known to possess anti-coagulant activity with acute toxicity. In this paper, we report a study on the interaction of bromadiolone with the plasma proteins bovine serum albumin (BSA) and human serum albumin (HSA), using the intrinsic fluorescence emission properties of bromadiolone. Bromadiolone is weakly fluorescent in aqueous buffer medium, with an emission at 397 nm. Binding of bromadiolone with serum albumins (SA) leads to a marked enhancement in the fluorescence emission intensity and steady state fluorescence anisotropy (r(ss)), accompanied by a blueshift of 10 nm. In the serum albumin-bromadiolone complex, selective excitation of tryptophan (Trp) residue results in emission from bromadiolone, thereby indicating a Förster type energy transfer from Trp to BRD. This quenching of Trp fluorescence by BRD was used to estimate the binding constant of the SA-BRD complex. The binding constants for BRD with BSA and HSA were 7.5 x 10(4) and 3.7 x 10(5)L mol(-1), respectively. Based on this, a new method involving SA as fluorescence-enhancing reagent for estimation of BRD in aqueous samples has been suggested. The detection limits of bromadiolone under the optimum conditions were 0.77 and 0.19 microg mL(-1) in presence of BSA and HSA, respectively. PMID:15925260

  17. Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling.

    PubMed

    Neacsu, Maria Victoria; Matei, Iulia; Micutz, Marin; Staicu, Teodora; Precupas, Aurica; Popa, Vlad Tudor; Salifoglou, Athanasios; Ionita, Gabriela

    2016-05-12

    The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements, and differential scanning microcalorimetry (μDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational changes to albumin if it is organized in a micellar form, while albumin influences the self-assembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The μDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the

  18. Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: Multi-spectroscopic and molecular modeling approaches

    NASA Astrophysics Data System (ADS)

    Zohoorian-Abootorabi, Toktam; Sanee, Hamideh; Iranfar, Hediyeh; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2012-03-01

    This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.

  19. A study of the adsorption of the amphiphilic penicillins cloxacillin and dicloxacillin onto human serum albumin using surface tension isotherms

    NASA Astrophysics Data System (ADS)

    Barbosa, Silvia; Leis, David; Taboada, Pablo; Attwood, David; Mosquera, Victor

    The interaction of human serum albumin (HSA) with two structurally similar anionic amphiphilic penicillins, cloxacillin and dicloxacillin, at 25°C has been examined by surface tension measurements under conditions at which the HSA molecule was positively (pH 4.5) or negatively charged (pH 7.4). Measurements were at fixed HSA concentrations (0.0125 and 0.125% w/v) and at drug concentrations over a range including, where possible, the critical micelle concentration (cmc). Interaction between anionic drugs and positively charged HSA at pH 7.4 resulted in an increase of the cmc of each drug as a consequence of its removal from solution by adsorption. Limited data for cloxacillin at pH 4.5 indicated an apparent decrease of the cmc in the presence of HSA suggesting a facilitation of the aggregation by association with the protein. Changes in the surface tension-log (drug concentration) plots in the presence of HSA have been discussed in terms of the adsorption of drug at the air-solution and protein-solution interfaces. Standard free energy changes associated with the micellization of both drugs and their adsorption at the air-solution interface have been calculated and compared.

  20. Regarding the effect that different hydrocarbon/fluorocarbon surfactant mixtures have on their complexation with HSA.

    PubMed

    Blanco, Elena; Messina, Paula; Ruso, Juan M; Prieto, Gerardo; Sarmiento, Félix

    2006-06-15

    The complexations between human serum albumin (HSA) and the sodium perfluorooctanoate/sodium octanoate and sodium perfluorooctanoate/sodium dodecanoate systems have been studied by a combination of electrical conductivity, ion-selective electrode, electrophoresis, and spectroscopy measurements. The binary mixtures of the surfactants deviated slightly from ideality. Binding plots revealed the existence of two specific binding sites, the first site being more accessible than the second. Positive cooperative binding has been found, thus revealing the importance of the hydrophobic interactions in both kinds of surfactants. The Gibbs energies of binding per mole of surfactant (DeltaG(nu)) were calculated from the Wyman binding potential where, on the basis of the elevated number of binding sites, a statistical contribution has been included. Initially these energies are large and negative, gradually decreasing as saturation is approached. Changes in the slope of Gibbs energies have been identified with the saturation of the first binding set. These facts denote that the surfactants under study have different favorite adsorption sites along the protein and that the adsorption process of perfluorooctanoate is more closely followed by dodecanoate than by octanoate. Finally, electrophoresis and spectroscopy measurements suggest induced conformational changes on HSA depending on the surfactant mixture as well as the mixed ratio.

  1. Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking.

    PubMed

    Kabir, Md Zahirul; Feroz, Shevin R; Mukarram, Abdul Kadir; Alias, Zazali; Mohamad, Saharuddin B; Tayyab, Saad

    2016-08-01

    Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.

  2. Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

    PubMed Central

    Yamauchi, Y; Litwin, A; Adams, L; Zimmer, H; Hess, E V

    1975-01-01

    The antihypertensive drug hydralazine can induce in man a syndrome similar to spontaneous systemic lupus erythematosus (SLE). The pathogenesis of this drug-induced syndrome is not understood. In this investigation, five groups of rabbits were studied: group I, 10 rabbits hyperimmunized with hydralazine conjugated to human serum albumin (HSA) in complete Freund's adjuvant (CFA); group II, four rabbits with HSA in CFA; group III, four rabbits with CFA alone; group IV, five rabbits with hydralazine conjugated to rabbit serum albumin (RSA); and group V, four rabbits with a major metabolite of hydralazine conjugated to HSA. The rabbits immunized with hydralazine-HSA developed rising titers of antibodies to hydralazine and progressively increasing amounts of antibodies to both single-stranded and native DNA. The antibodies to DNA were cross-reactive with hydralazine as determined by inhibition of DNA binding and DNA hemagglutination tests. Similar results were obtained in rabbits immunized with the metabolite-HSA compound except the major hapten antibody response was to the metabolite. The DNA antibodies in this group were also capable of being absorbed by metabolite-HSA as well as hydralazine-HSA, indicative of the cross-reactivity between hydralazine and its metabolite. Immunization with hydralazine-RSA caused rabbits to produce antibodies to hydralazine but not to DNA, indicating the requirement for an immune response to the carrier protein in order for antibodies reactive with DNA to be produced. Thus, hyperimmunization of rabbits with hydralazine-protein conjugates may provide a useful animal model of SLE. The data suggests that an immune response to hydralazine may be important in human hydralazine-induced SLE. Images PMID:808562

  3. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway

    PubMed Central

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to 125I-albumin. HMGB1 induced an increase in 125I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  4. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway.

    PubMed

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to (125)I-albumin. HMGB1 induced an increase in (125)I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  5. Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole

    NASA Astrophysics Data System (ADS)

    Punith, Reeta; Seetharamappa, J.

    2012-06-01

    The present study employed different optical spectroscopic techniques viz., fluorescence, FTIR, circular dichroism (CD) and UV-vis absorption spectroscopy to investigate the mechanism of interaction of an anticancer drug, anastrozole (AZ) with transport proteins viz., bovine serum albumin (BSA) and human serum albumin (HSA). The drug, AZ quenched the intrinsic fluorescence of protein and the analysis of results revealed the presence of dynamic quenching mechanism. The binding characteristics of drug-protein were computed. The thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be +92.99 kJ/mol and +159.18 J/mol/K for AZ-BSA and, +99.43 kJ/mol and +159.19 J/mol/K for AZ-HSA, respectively. These results indicated that the hydrophobic forces stabilized the interaction between the drug and protein. CD, FTIR, absorption, synchronous and 3D fluorescence results indicated that the binding of AZ to protein induced structural perturbation in both serum albumins. The distance, r between the drug and protein was calculated based on the theory of Förster's resonance energy transfer and found to be 5.9 and 6.24 nm, respectively for AZ-BSA and AZ-HSA.

  6. [Preparation of Human Serum Albumin Micro/Nanotubes].

    PubMed

    Jiao, Pei-pei; Guo, Yan-li; Niu, Ai-hua; Kang, Xiao-feng

    2016-01-01

    In this research, protein micro/nanotubes were fabricated by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethyleneimine (PEI) into polycarbonate (PC) membranes. The experimental conditions of pH values, ionic strength, the depositions cycles and the diameter of porous membrane were discussed. The morphology and composition of tubes were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS). The results show that pH and ionic strength of the solution are the key factors that influence the effect of assembly. Micro/nanotubes with good opening hollow tubular structure were obtained when pH 7.4 HSA solution and pH 10.3 PEI solution without NaCl were used in synthesis procedure. The outer diameter of tube was dependent on the PC template, thus the micro/nanotubes size was controlled by the wall thickness, which can be adjusted by the number of layers of the HSA and PEI deposited along the pore walls. To avoid the thin wall being damaged in dissolving the template and vacuum drying, the PEI/HSA bilayer number should not be less than 3. The polar solvent N,N-dimethylformamide (DMF) can dissolve PC template to release the micro/nanotubes. PMID:27228766

  7. [Preparation of Human Serum Albumin Micro/Nanotubes].

    PubMed

    Jiao, Pei-pei; Guo, Yan-li; Niu, Ai-hua; Kang, Xiao-feng

    2016-01-01

    In this research, protein micro/nanotubes were fabricated by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethyleneimine (PEI) into polycarbonate (PC) membranes. The experimental conditions of pH values, ionic strength, the depositions cycles and the diameter of porous membrane were discussed. The morphology and composition of tubes were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS). The results show that pH and ionic strength of the solution are the key factors that influence the effect of assembly. Micro/nanotubes with good opening hollow tubular structure were obtained when pH 7.4 HSA solution and pH 10.3 PEI solution without NaCl were used in synthesis procedure. The outer diameter of tube was dependent on the PC template, thus the micro/nanotubes size was controlled by the wall thickness, which can be adjusted by the number of layers of the HSA and PEI deposited along the pore walls. To avoid the thin wall being damaged in dissolving the template and vacuum drying, the PEI/HSA bilayer number should not be less than 3. The polar solvent N,N-dimethylformamide (DMF) can dissolve PC template to release the micro/nanotubes.

  8. Conformational and adsorptive characteristics of albumin affect interfacial protein boundary lubrication: from experimental to molecular dynamics simulation approaches.

    PubMed

    Fang, Hsu-Wei; Hsieh, Man-Ching; Huang, Huei-Ting; Tsai, Cheng-Yen; Chang, Min-Hui

    2009-02-01

    The lifetime of artificial joints is mainly determined by their biotribological properties. Synovial fluid which consists of various biological molecules acts as the lubricant. Among the compositions of synovial fluid, albumin is the most abundant protein. Under high load and low sliding speed articulation of artificial joint, it is believed the lubricants form protective layers on the sliding surfaces under the boundary lubrication mechanism. The protective molecular layer keeps two surfaces from direct collision and thus decreases the possibility of wear damage. However, the lubricating ability of the molecular layer may vary due to the conformational change of albumin in the process. In this study, we investigated the influence of albumin conformation on the adsorption behaviors on the articulating surfaces and discuss the relationship between adsorbed albumin and its tribological behaviors. We performed the friction tests to study the effects of albumin unfolding on the frictional behaviors. The novelty of this research is to further carry out molecular dynamics simulation, and protein adsorption experiments to investigate the mechanisms of the albumin-mediated boundary lubrication of arthroplastic materials. It was observed that the thermal processes induce the loss of secondary structure of albumin. The compactness of the unfolded structure leads to a higher adsorption rate onto the articulating material surface and results in the increase of friction coefficient.

  9. Conformational changes in human serum albumin induced by sodium perfluorooctanoate in aqueous solutions.

    PubMed

    Messina, Paula V; Prieto, Gerardo; Ruso, Juan M; Sarmiento, Félix

    2005-08-18

    Conformational changes in the bulk solution and at the air-aqueous interface of human serum albumin (HSA) induced by changes in concentration of sodium perfluorooctanoate (C(7)F(15)COO(-)Na(+)) were studied by difference spectroscopy, zeta-potential data, and axisymmetric drop shape analysis. zeta-potential was used to monitor the formation of the HSA-C(7)F(15)COO(-)Na(+) complex and the surface charge of the complex. The conformational transition of HSA in the bulk solution was followed as a function of denaturant concentration by absorbance measurements at 280 nm. The data were analyzed to obtain values for the Gibbs energies of the transition in water (DeltaG(0)(W)) and in a hydrophobic environment (DeltaG(0)(hc)) pertaining to saturated protein-surfactant complexes. The conformational changes that surfactants induce in HSA molecules alter its absorption behavior at the air-water interface. Dynamic surface measurements were used to evaluate this behavior. At low [C(7)F(15)COO(-)Na(+)], proteins present three adsorption regimes: induction time, monolayer saturation, and interfacial gelation. When surfactant concentration increases and conformational changes in the bulk solution occur, the adsorption regimes disappear. HSA molecules in an intermediate conformational state migrate to the air-water interface and form a unique monolayer. At high [C(7)F(15)COO(-)Na(+)], the adsorption of denatured molecules exhibits a behavior analogous to that of dilute solutions.

  10. Human albumin solution for patients with cirrhosis and acute on chronic liver failure: Beyond simple volume expansion

    PubMed Central

    Valerio, Christopher; Theocharidou, Eleni; Davenport, Andrew; Agarwal, Banwari

    2016-01-01

    To provide an overview of the properties of human serum albumin (HSA), and to review the evidence for the use of human albumin solution (HAS) in critical illness, sepsis and cirrhosis. A MEDLINE search was performed using the terms “human albumin”, “critical illness”, “sepsis” and “cirrhosis”. The references of retrieved articles were reviewed manually. Studies published between 1980 and 2014 were selected based on quality criteria. Data extraction was performed by all authors. HSA is the main plasma protein contributing greatly to its oncotic pressure. HSA demonstrates important binding properties for endogenous and exogenous toxins, drugs and drug metabolites that account for its anti-oxidant and anti-inflammatory properties. In disease states, hypoalbuminaemia is secondary to decreased HSA production, increased loss or transcapillary leakage into the interstitial space. HSA function can be also altered in disease with reduced albumin binding capacity and increased production of modified isoforms. HAS has been used as volume expander in critical illness, but received criticism due to cost and concerns regarding safety. More recent studies confirmed the safety of HAS, but failed to show any survival benefit compared to the cheaper crystalloid fluids, therefore limiting its use. On the contrary, in cirrhosis there is robust data to support the efficacy of HAS for the prevention of circulatory dysfunction post-large volume paracentesis and in the context of spontaneous bacterial peritonitis, and for the treatment of hepato-renal syndrome and hypervolaemic hyponatraemia. It is likely that not only the oncotic properties of HAS are beneficial in cirrhosis, but also its functional properties, as HAS replaces the dysfunctional HSA. The role of HAS as the resuscitation fluid of choice in critically ill patients with cirrhosis, beyond the established indications for HAS use, should be addressed in future studies. PMID:26981172

  11. Evidence for Water-Tuned Structural Differences in Proteins: An Approach Emphasizing Variations in Local Hydrophilicity

    PubMed Central

    Hinderberger, Dariush

    2012-01-01

    We present experimental evidence for the significant effect that water can have on the functional structure of proteins in solution. Human (HSA) and Bovine Serum Albumin (BSA) have an amino acid sequence identity of 75.52% and are chosen as model proteins. We employ EPR-based nanoscale distance measurements using double electron-electron resonance (DEER) spectroscopy and both albumins loaded with long chain fatty acids (FAs) in solution to globally (yet indirectly) characterize the tertiary protein structures from the bound ligands’ points of view. The complete primary structures and crystal structures of HSA and as of recently also BSA are available. We complement the picture as we have recently determined the DEER-derived solution structure of HSA and here present the corresponding BSA solution structure. The characteristic asymmetric FA distribution in the crystal structure of HSA can surprisingly be observed by DEER in BSA in solution. This indicates that the BSA conformational ensemble in solution seems to be narrow and close to the crystal structure of HSA. In contrast, for HSA in solution a much more symmetric FA distribution was found. Thus, conformational adaptability and flexibility dominate in the HSA solution structure while BSA seems to lack these properties. We further show that differences in amino acid hydropathies of specific structural regions in both proteins can be used to correlate the observed difference in the global (tertiary) solution structures with the differences on the primary structure level. PMID:23049837

  12. Characterisation of interaction between food colourant allura red AC and human serum albumin: multispectroscopic analyses and docking simulations.

    PubMed

    Wu, Di; Yan, Jin; Wang, Jing; Wang, Qing; Li, Hui

    2015-03-01

    Binding interaction of human serum albumin (HSA) with allura red AC, a food colourant, was investigated at the molecular level through fluorescence, ultraviolet-visible, circular dichroism (CD) and Raman spectroscopies, as well as protein-ligand docking studies to better understand the chemical absorption, distribution and transportation of colourants. Results show that allura red AC has the ability to quench the intrinsic fluorescence of HSA through static quenching. The negative values of the thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bond and van der Waals forces are dominant in the binding between the food colourant and HSA. The CD and Raman spectra showed that the binding of allura red AC to HSA induces the rearrangement of the carbonyl hydrogen-bonding network of polypeptides, which changes the HSA secondary structure. This colourant is bound to HSA in site I, and the binding mode was further analysed with the use of the CDOCKER algorithm in Discovery Studio.

  13. Multifunctional Effect of Human Serum Albumin Reduces Alzheimer's Disease Related Pathologies in the 3xTg Mouse Model.

    PubMed

    Ezra, Assaf; Rabinovich-Nikitin, Inna; Rabinovich-Toidman, Polina; Solomon, Beka

    2015-01-01

    Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifests simultaneously, eventually leading to cognitive impairment and death. No treatment is currently available; however, an agent addressing several key pathologies simultaneously has a better therapeutic potential. Human serum albumin (HSA) is a highly versatile protein, harboring multifunctional properties that are relevant to key pathologies underlying AD. This study provides insight into the mechanism for HSA's therapeutic effect. In vivo, a myriad of beneficial effects were observed by pumps infusing HSA intracerebroventricularly, for the first time in an AD 3xTg mice model. A significant effect on amyloid-β (Aβ) pathology was observed. Aβ1-42, soluble oligomers, and total plaque area were reduced. Neuroblastoma SHSY5Y cell line confirmed that the reduction in Aβ1-42 toxicity was due to direct binding rather than other properties of HSA. Total and hyperphosphorylated tau were reduced along with an increase in tubulin, suggesting increased microtubule stability. HSA treatment also reduced brain inflammation, affecting both astrocytes and microglia markers. Finally, evidence for blood-brain barrier and myelin integrity repair was observed. These multidimensional beneficial effects of intracranial administrated HSA, together or individually, contributed to an improvement in cognitive tests, suggesting a non-immune or Aβ efflux dependent means for treating AD.

  14. Structure of the methyl orange-binding site on human serum albumin and its color-change mechanism.

    PubMed

    Ito, Shigenori; Yamamoto, Daisuke

    2015-01-01

    The goal in this study was to clarify the color-change mechanisms of methyl orange (MO) bound to human serum albumin (HSA) and the structure of the binding site. The absorbance of the MOHSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pH-dependent experimental data were consistent with the data calculated using the Henderson-Hasselbalch equation and pKa values (3.8, MO; 1.4, carboxyl group). The extent of the binding of MO to an HSA molecule was determined to be 1-4 by performing surface plasmon resonance analysis. Furthermore, the binding of MO to HSA was inhibited by warfarin. A fitting model of MO to HSA was created to evaluate these results based on PDB data (warfarin-HSA complex: 2BXD) and protein-structure analysis. The color-change mechanism of the MO-HSA complex appears to be as follows: the dissociated sulfo group of MO binds to Arg218/Lys444 sidechains through electrostatic interaction in the warfarin-binding site, and, subsequently, the color change occurs through a proton exchange between the diazenyl group and the γ-carboxyl group of Glu292. The color-changed MO is fixed in the warfarin-binding site. These results could support the development of a reliable dye-binding method and of a new method for staining diverse tissues that is based on a validated mechanism.

  15. Synthesis and characterization of protein and polylysine conjugates of sulfamethoxazole and sulfanilic acid for investigation of sulfonamide drug allergy.

    PubMed

    Tatake, J G; Knapp, M M; Ressler, C

    1991-01-01

    Conjugates of sulfamethoxazole (SMX) with human serum albumin (HSA), transferrin (TR), and poly(L-lysine) (PL, degrees of polymerization 16 and 430) have been prepared. As a model, succinylSMX-glycine methyl ester was synthesized by carbodiimide and active ester routes. The proteins and PL were acylated with succinylSMX succinimido ester, affording conjugates (succinylSMX)2-21-HSA, (succinylSMX)17,27-TR, (succinylSMX)11-Lys16, and (succinylSMX)71-Lys430 in which SMX was linked by a spacer chain of four carbons. This represents substitution of up to 35, 46, 65, and 17% of the amino groups of HSA, TR, PL16, and PL430, respectively. HSA was also acylated with the succinimido esters of succinylSMX-glycine and succinylSMX-epsilon-aminohexanoic acid, affording conjugates (succinylSMX-Gly)53-HSA and (succinylSMX-epsilon-NH2hex)51-HSA. In these conjugates SMX was linked by a spacer chain of 7 and 11 carbons, respectively, and almost all the amino groups of HSA were substituted. Factors apparently influencing the extent of conjugation to HSA were the stability of the active ester and the solubility of the conjugation reaction mixture. A sulfanilic acid (SA) conjugate, containing 12 mol of ligand/mol of HSA, was also prepared. The route of synthesis involved acylation of HSA with sulfanilyl fluoride. N-epsilon-Sulfanilyl-L-lysine dihydrochloride, required for quantitation of bound SA, was synthesized by a new route starting from alpha-Boc-L-lysine. Conjugates (sulfanilyl)12-HSA and (succinylSMX)13-HSA, differing in molecular weight from HSA by only 2.6 and 6.5%, were distinguishable from HSA by gel-filtration HPLC, as were the more highly substituted conjugates from their respective unsubstituted materials.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins.

    PubMed

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  17. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins

    PubMed Central

    Nusrat, Saima; Siddiqi, Mohammad Khursheed; Zaman, Masihuz; Zaidi, Nida; Ajmal, Mohammad Rehan; Alam, Parvez; Qadeer, Atiyatul; Abdelhameed, Ali Saber

    2016-01-01

    Exogenous drugs that are used as antidote against chemotheray, inflammation or viral infection, gets absorbed and interacts reversibly to the major serum transport protein i.e. albumins, upon entering the circulatory system. To have a structural guideline in the rational drug designing and in the synthesis of drugs with greater efficacy, the binding mechanism of an antineoplastic and anti-inflammatory drug Nordihydroguaiaretic acid (NDGA) with human and bovine serum albumins (HSA & BSA) were examined by spectroscopic and computational methods. NDGA binds to site II of HSA with binding constant (Kb) ~105 M-1 and free energy (ΔG) ~ -7.5 kcal.mol-1. It also binds at site II of BSA but with lesser binding affinity (Kb) ~105 M-1 and ΔG ~ -6.5 kcal.mol-1. The negative value of ΔG, ΔH and ΔS for both the albumins at three different temperatures confirmed that the complex formation process between albumins and NDGA is spontaneous and exothermic. Furthermore, hydrogen bonds and hydrophobic interactions are the main forces involved in complex formation of NDGA with both the albumins as evaluated from fluorescence and molecular docking results. Binding of NDGA to both the albumins alter the conformation and causes minor change in the secondary structure of proteins as indicated by the CD spectra. PMID:27391941

  18. Interaction of bovine serum albumin protein with self assembled monolayer of mercaptoundecanoic acid

    NASA Astrophysics Data System (ADS)

    Poonia, Monika; Agarwal, Hitesh; Manjuladevi, V.; Gupta, R. K.

    2016-05-01

    Detection of proteins and other biomolecules in liquid phase is the essence for the design of a biosensor. The sensitivity of a sensor can be enhanced by the appropriate functionalization of the sensing area so as to establish the molecular specific interaction. In the present work, we have studied the interaction of bovine serum albumin (BSA) protein with a chemically functionalized surface using a quartz crystal microbalance (QCM). The gold-coated quartz crystals (AT-cut/5 MHz) were functionalized by forming self-assembled monolayer (SAM) of 11-Mercaptoundecanoic acid (MUA). The adsorption characteristics of BSA onto SAM of MUA on quartz crystal are reported. BSA showed the highest affinity for SAM of MUA as compared to pure gold surface. The SAM of MUA provides carboxylated surface which enhances not only the adsorption of the BSA protein but also a very stable BSA-MUA complex in the liquid phase.

  19. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  20. Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

    PubMed

    Yang, Byungseop; Lim, Sung In; Kim, Jong Chul; Tae, Giyoong; Kwon, Inchan

    2016-05-01

    Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG. PMID:27050863

  1. Revealing deposition mechanism of colloid particles on human serum albumin monolayers.

    PubMed

    Nattich-Rak, Małgorzata; Adamczyk, Zbigniew; Kujda, Marta

    2016-01-01

    Colloid particle deposition was applied in order to characterize human serum albumin (HSA) monolayers on mica adsorbed under diffusion transport at pH 3.5. The surface concentration of HSA was determined by a direct AFM imaging of single molecules. The electrokinetic characteristics of the monolayers for various ionic strength were done by in situ streaming potential measurements. In this way the mean-field zeta potential of monolayers was determined. It was shown that the initially negative potential changed its sign for HSA surface concentrations above 2800μm(-2) that was interpreted as overcharging effect. The monolayers were also characterized by the colloid deposition method where negatively charged polystyrene particles, 810nm in diameter were used. The kinetics of particle deposition and their maximum coverage were determined as a function of the HSA monolayer surface concentration. An anomalous deposition of particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field theoretical predictions. This effect was quantitatively interpreted in terms of the random site sequential adsorption model. It was shown that efficient immobilization of particles only occurs at adsorption sites formed by three and more closely adsorbed HSA molecules. These results can be exploited as useful reference data for the analysis of deposition phenomena of bioparticles at protein monolayers that has practical significance for the regulation of the bioadhesive properties of surfaces.

  2. Molecular interaction and energy transfer between human serum albumin and bioactive component Aloe dihydrocoumarin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Li, Lin; Tang, Ya-Lin

    2008-10-01

    Aloe dihydrocoumarin is an antioxidant and a candidate of immunomodulatory drug on the immune system and can balance physiological reactive oxygen species (ROS) levels which may be useful to maintain homeostasis. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydrocoumarin with human serum albumin (HSA) has been investigated by fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydrocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. A Förster type fluorescence resonance energy transfer mechanism is involved in this quenching of Trp fluorescence by Aloe dihydrocoumarin. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydrocoumarin with HSA causes a conformational change of the protein, with the loss of α-helix stability and the gain of β-sheet and β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FT-IR experiments along with the docking studies suggest that Aloe dihydrocoumarin binds to residues located in subdomain IIA of HSA.

  3. Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin.

    PubMed

    Meng, Xiaoli; Maggs, James L; Usui, Toru; Whitaker, Paul; French, Neil S; Naisbitt, Dean J; Park, B Kevin

    2015-01-20

    Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.

  4. Albumin and multiple sclerosis.

    PubMed

    LeVine, Steven M

    2016-04-12

    Leakage of the blood-brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth.

  5. Albumin and multiple sclerosis.

    PubMed

    LeVine, Steven M

    2016-01-01

    Leakage of the blood-brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth. PMID:27067000

  6. Novel 7-(dimethylamino)fluorene-based fluorescent probes and their binding to human serum albumin.

    PubMed

    Park, Kwanghee Koh; Park, Joon Woo; Hamilton, Andrew D

    2009-10-21

    A novel solvatochromic fluorescent molecule, 9,9-dibutyl-7-(dimethylamino)-2-fluorenesulfonate 2 was synthesized from 2-nitrofluorene in moderate yield. The fluorescence spectra of 2 and 7-(dimethylamino)-2-fluorenesulfonate 1 shift to shorter wavelengths as the polarity of the medium decreases. Both 1 and 2 bind to hydrophobic sites of human serum albumin (HSA). The apparent binding constants were determined by fluorescence titration to be 0.37 x 10(6) M(-1) for 1 and 2.2 x 10(6) M(-1) for 2. The energy of the Trp-214 fluorescence of HSA is transferred to the HSA-bound fluorophores with near 100% efficiency. The covalent bonding of acrylodan (AC) to Cys-34 has little effect on the binding affinity of 2 to HSA or fluorescent behavior of HSA-bound 2. Bound 2 also has little effect on the fluorescence of AC, but 2-->AC and Trp-214-->2-->AC resonance energy transfers were observed. Competitive binding between the fluorene compounds and other ligands such as 1-anilino-8-naphthalenesulfonate, aspirin, S-(+)-ibuprofen and phenylbutazone were also studied fluorometrically. The results indicated that the primary binding site of 2 to HSA is site II in domain IIIA, whereas 1 binds to site I in domain IIA, but a different region from the phenylbutazone binding site. Because of its large molar absorptivity, strong fluorescence, sensitivity to its environment, and high binding constant to HSA, 2 can be used successfully in the study of proteins and their binding properties. PMID:19795061

  7. Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

    PubMed

    Santra, Manas Kumar; Banerjee, Abhijit; Krishnakumar, Shyam Sundar; Rahaman, Obaidur; Panda, Dulal

    2004-05-01

    The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

  8. 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.

  9. [Analysis of FT-IR-ATR spectra of serum proteins adsorbed on carbonaceous materials].

    PubMed

    Li, Bogang; Na, Juanjuan; Yin, Jie; Yin, Guangfu

    2006-10-01

    To clarify the reason causing difference of serum proteins adsorbability on different carbonaceous materials, FT-IR-ATR spectra of human serum albumin (HSA) and human serum fibrinogen(HFG) before and after adsorbing on diamond like carbon film (DLC),diamond film (DF) and graphite were analyzed. It has been shown that there are hydrogen bond because of -NH at the interfaces of HSA-DLC, HFG-DF and HFG-graphite. Based on the results, earlier research conclusion that the adsorbability of HSA on DLC higher than that on DF and graphite, but on DF and graphite the adsorption of HFG takes precedence can be explained rationally.

  10. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  11. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  12. Water-Soluble alpha,beta-unsaturated aldehydes of cigarette smoke induce carbonylation of human serum albumin.

    PubMed

    Colombo, Graziano; Aldini, Giancarlo; Orioli, Marica; Giustarini, Daniela; Gornati, Rosalba; Rossi, Ranieri; Colombo, Roberto; Carini, Marina; Milzani, Aldo; Dalle-Donne, Isabella

    2010-03-01

    Cigarette smoking is a major risk factor for developing pulmonary and cardiovascular diseases as well as some forms of cancer. Understanding the mechanisms by which smoking contributes to disease remains a major research focus. Increased levels of carbonylated serum proteins are present in smokers; albumin is the major carbonylated protein in the bronchoalveolar lavage fluid of older smokers. We have investigated the susceptibility of human serum albumin (HSA) to alpha,beta-unsaturated aldehyde-induced carbonylation when exposed to whole-phase cigarette smoke extract (CSE). Fluorescence studies with fluorescent probes showed depletion of HSA Cys34 free thiol and marked decrease of free Lys residues. Spectrophotometric and immunochemical carbonyl assays after carbonyl derivatization with 2,4-dinitrophenylhydrazine revealed the formation of covalent carbonyl adducts. Nanoscale capillary liquid chromatography and electrospray tandem mass spectrometry analysis detected acrolein and crotonaldehyde Michael adducts at Cys34, Lys525, Lys351, and His39 at all the CSE concentrations tested. Lys541 and Lys545 were also found to form a Schiff base with acrolein. The carbonyl scavenger drugs, hydralazine and pyridoxamine, partially prevented CSE-induced HSA carbonylation. Carbonylation of HSA associated with cigarette smoking might result in modifications of its antioxidant properties and transport functions of both endogenous and exogenous compounds.

  13. Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration

    PubMed Central

    Ebrahem, Quteba; Renganathan, Kutralanathan; Sears, Jonathan; Vasanji, Amit; Gu, Xiaorong; Lu, Liang; Salomon, Robert G.; Crabb, John W.; Anand-Apte, Bela

    2006-01-01

    Choroidal neovascularization (CNV), the advanced stage of age-related macular degeneration (AMD), accounts for >80% of vision loss in AMD. Carboxyethylpyrrole (CEP) protein modifications, uniquely generated from oxidation of docosahexaenoate-containing lipids, are more abundant in Bruch’s membrane from AMD eyes. We tested the hypothesis that CEP protein adducts stimulate angiogenesis and possibly contribute to CNV in AMD. Human serum albumin (HSA) or acetyl-Gly-Lys-O-methyl ester (dipeptide) were chemically modified to yield CEP-modified HSA (CEP-HSA) or CEP-dipeptide. The in vivo angiogenic properties of CEP-HSA and CEP-dipeptide were demonstrated by using the chick chorioallantoic membrane and rat corneal micropocket assays. Low picomole amounts of CEP-HSA and CEP-dipeptide stimulated neovascularization. Monoclonal anti-CEP antibody neutralized limbal vessel growth stimulated by CEP-HSA, whereas anti-VEGF antibody was found to only partially neutralize vessel growth. Subretinal injections of CEP-modified mouse serum albumin exacerbated laser-induced CNV in mice. In vitro treatments of human retinal pigment epithelial cells with CEP-dipeptide or CEP-HSA did not induce increased VEGF secretion. Overall, these results suggest that CEP-induced angiogenesis utilizes VEGF-independent pathways and that anti-CEP therapeutic modalities might be of value in limiting CNV in AMD. PMID:16938854

  14. Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-07-01

    In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461

  15. Immunotoxicity Assessment of Rice-Derived Recombinant Human Serum Albumin Using Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Fu, Kai; Cheng, Qin; Liu, Zhenwei; Chen, Zhen; Wang, Yan; Ruan, Honggang; Zhou, Lu; Xiong, Jie; Xiao, Ruijing; Liu, Shengwu; Zhang, Qiuping; Yang, Daichang

    2014-01-01

    Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4+ and CD8+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development. PMID:25099245

  16. Binding of Breviscapine Toward Serum Albumin Studied by Spectroscopic and Electrochemical Techniques.

    PubMed

    Liu, Wei; Chen, Yaqing; Chen, Hui; Zhang, Ying

    2016-09-01

    Breviscapine, a cerebrovascular drugs extracted from the Chinese herb Erigeron breviscapinus, has been frequently used to clinically treat cerebrovascular diseases such as cerebral thrombosis, cerebral infarction, and cerebral circulation insufficiency. In order to understand its pharmacology or toxicity, the binding mechanism of breviscapine to a model protein, human serum albumin (HSA), was probed by fluorescence, circular dichroism, Fourier transform infrared spectroscopy (FTIR), and electrochemical impedance spectroscopy approaches. The binding affinities and number of the drug with HSA were about 1.73 × 10(4)  M(-1) and 0.99 at 293 K, respectively. The conformation of the protein was slightly altered after interacting with breviscapine. The drug-protein complex was mainly stabilized by electrostatic forces.

  17. Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Raut, Sangram; Chib, Rahul; Butler, Susan; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2014-09-01

    This work reports on the chromophores interactions within protein-protected gold nanoclusters. We conducted spectroscopic studies of fluorescence emissions originated from gold nanoclusters and intrinsic tryptophan (Trp) in BSA or HSA proteins. Both steady state fluorescence and lifetime measurements showed a significant Forster Resonance Energy Transfer (FRET) from Trp to the gold nanocluster. Tryptophan lifetimes in the case of protein-protected gold nanoclusters are 2.6 ns and 2.3 ns for BSA and HSA Au clusters while 5.8 ns for native BSA and 5.6 for native HSA. The apparent distances from Trp to gold nanocluster emission center, we estimated as 24.75 Å for BSA and 23.80 Å for HSA. We also studied a potassium iodide (KI) quenching of protein-protected gold nanoclusters and compared with the quenching of BSA and HSA alone. The rates of Trp quenching were smaller in BSA-Au and HSA-Au nanoclusters than in the case of free proteins, which is consistent with shorter lifetime of quenched Trp(s) and lower accessibility for KI. While Trp residues were quenched by KI, the emissions originated from nanoclusters were practically unquenched. In summary, for BSA and HSA Au clusters, we found 55% and 59% energy transfer efficiency respectively from tryoptophan to gold clusters. We believe this interaction can be used to our advantage in terms of developing resonance energy transfer based sensing applications.

  18. Interaction of weakly bound antibiotics neomycin and lincomycin with bovine and human serum albumin: biophysical approach.

    PubMed

    Keswani, Neelam; Choudhary, Sinjan; Kishore, Nand

    2010-07-01

    The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UV-visible, steady state and time resolved fluorescence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van't Hoff enthalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at different ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the predominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of lincomycin with these proteins.

  19. Adsorption of proteins at physiological concentrations on pegylated surfaces and the compatibilizing role of adsorbed albumin with respect to other proteins according to optical waveguide lightmode spectroscopy (OWLS).

    PubMed

    Leclercq, Laurent; Modena, Enrico; Vert, Michel

    2013-01-01

    In literature, contacts between pegylated compounds and blood proteins are generally discussed in terms of excluded volume-related repulsions although adsorption and compatibility have been reported for some of these proteins occasionally. The major problem to investigate the behavior of blood in contact with pegylated surfaces is the complexity of the medium and especially the presence of albumin in large excess. In a model approach, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the fate of albumin, fibrinogen, and γ-globulins at physiological concentrations in pH = 7.4 isotonic HEPES buffer after contact with SiTiO2 chips coated with diblock poly(DL-lactic acid)-block-poly(ethylene oxide)s and triblock poly(DL-lactic acid)-block-poly(ethylene oxide)-block-poly(DL-lactic acid) copolymers. Corresponding homopolymers were used as controls. The three protein systems were investigated separately, as a mixture and when added successively according to different orders of addition. OWLS gave access to the mass and the thickness of adhering protein layers that resist washing with HEPES buffer. Protein depositions were detected regardless of the presence of poly(ethylene glycol) segments on surfaces. Adsorption depended on the protein, on the surface and also on the presence of the other proteins. Unexpectedly any surface coated with a layer of adsorbed albumin prevented deposition of other proteins, including albumin itself. This outstanding finding suggests that it was the presence of albumin adsorbed on a surface, pegylated or not, that made that surface compatible with other proteins. As a consequence, dipping a device to be in contact with the blood of a patient in a solution of albumin could be a very simple means to avoid further protein deposition and maybe platelets adhesion after in vivo implantation.

  20. Role of Serum Interleukin 6, Albumin and C-Reactive Protein in COPD Patients

    PubMed Central

    Emami Ardestani, Mohammad

    2015-01-01

    Background: Chronic obstructive pulmonary disease (COPD) is a non-specific inflammation, which involves the airways, lung parenchyma and pulmonary vessels. The inflammation causes the activation of inflammatory cells and the release of various inflammatory mediators such as interleukin-8 (IL-8), IL-6 and tumor necoris factor alpha (TNF-a). The purpose of the present study was to measure serum IL-6, C-reactive protein (CRP) (as a positive phase reactant) and albumin level (as a negative phase reactant) in COPD patients (only due to cigarette smoking not bio-mass), non COPD smokers and healthy subjects using enzyme-linked immunosorbent assay (ELISA); we compared the differences in inflammatory factors among groups. Materials and Methods: A total of 180 males were enrolled in this study and divided into three equal groups. The first group was 60 smokers who had COPD. The second group included 60 smokers without COPD and the third group consisted of people who were not smokers and did not have COPD; 5 mL of venous blood was taken from all participants and it was collected in a test tube containing anticoagulant and then centrifuged at 3000 rpm for 10 minutes. Serum was separated and used to measure the amount of IL-6, CRP and albumin. Spirometry was performed according to the criteria set by the American Thoracic Society. Results: The mean serum level of IL-6 was 83.2±7.5 pg/mL in group I, 54.9±24.3 pg/mL in group II and 46.9±10.4 pg/mL in group III. There was a significant difference among the three groups (P<0.001). The mean serum level of CRP was 28.9±14.9 mg/dL in the first group, 19.9±8.5 mg/dL in the second group and 4.2±2.3 mg/dL in the third group (P=0.02). But by controlling the confounding effects of age, this difference was not significant (P=0.49). The mean serum level of albumin was I 4.1±0.57 mg/dL in group I, 4.3±0.56 mg/dL in group II and 4.1±0.53 mg/dL in group III. There was no significant difference among the three groups in this regard (P=0

  1. A Rapid Study of Botanical Drug-Drug Interaction with Protein by Re-ligand Fishing using Human Serum Albumin-Functionalized Magnetic Nanoparticles.

    PubMed

    Qing, Lin-Sen; Xue, Ying; Ding, Li-Sheng; Liu, Yi-Ming; Liang, Jian; Liao, Xun

    2015-12-01

    A great many active constituents of botanical drugs bind to human serum albumin (HSA) reversibly with a dynamic balance between the free- and bound-forms in blood. The curative or side effect of a drug depends on its free-form level, which is always influenced by other drugs, combined dosed or multi-constituents of botanical drugs. This paper presented a rapid and convenient methodology to investigate the drug-drug interactions with HSA. The interaction of two steroidal saponins, dioscin and pseudo-protodioscin, from a botanical drug was studied for their equilibrium time and equilibrium amount by re-ligand fishing using HSA functionalized magnetic nanoparticles. A clear competitive situation was obtained by this method. The equilibrium was reached soon about 15 s at a ratio of 0.44: 1. Furthermore, the interaction of pseudo-protodioscin to total steroidal saponins from DAXXK was also studied. The operation procedures of this method were faster and more convenient compared with other methods reported. PMID:26882690

  2. Human Serum Albumin Complexed with Myristate and AZT

    SciTech Connect

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong

    2008-06-16

    3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

  3. Developing Anticancer Copper(II) Pro-drugs Based on the Nature of Cancer Cells and the Human Serum Albumin Carrier IIA Subdomain.

    PubMed

    Gou, Yi; Qi, Jinxu; Ajayi, Joshua-Paul; Zhang, Yao; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2015-10-01

    To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1-C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

  4. Association of a high normalized protein catabolic rate and low serum albumin level with carpal tunnel syndrome in hemodialysis patients

    PubMed Central

    Huang, Wen-Hung; Hsu, Ching-Wei; Weng, Cheng-Hao; Yen, Tzung-Hai; Lin, Jui-Hsiang; Lee, Meng

    2016-01-01

    Abstract Carpal tunnel syndrome (CTS) is the most common mononeuropathy in patients with end-stage renal disease (ESRD). The association between chronic inflammation and CTS in hemodialysis (HD) patients has rarely been investigated. HD patients with a high normalized protein catabolic rate (nPCR) and low serum albumin level likely have adequate nutrition and inflammation. In this study, we assume that a low serum albumin level and high nPCR is associated with CTS in HD patients. We recruited 866 maintenance hemodialysis (MHD) patients and divided them into 4 groups according to their nPCR and serum albumin levels: (1) nPCR <1.2 g/kg/d and serum albumin level <4 g/dL; (2) nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL; (3) nPCR <1.2 g/kg/d and serum albumin level ≥4 g/dL; and (4) nPCR ≥1.2 g/kg/d and serum albumin level ≥4 g/dL. After adjustment for related variables, HD duration and nPCR ≥1.2 g/kg/d and serum albumin level <4 g/dL were positively correlated with CTS. By calculating the area under the receiver-operating characteristic curve, we calculated that the nPCR and HD duration cut-off points for obtaining the most favorable Youden index were 1.29 g/kg/d and 7.5 years, respectively. Advance multivariate logistic regression analysis revealed that in MHD patients, nPCR ≥1.29 g/kg/d and serum albumin <4 g/dL, and also HD duration >7.5 years were associated with CTS. A high nPCR and low serum albumin level, which likely reflect adequate nutrition and inflammation, were associated with CTS in MHD patients. PMID:27368039

  5. [Detection of autoantibodies to polymerized human serum albumin].

    PubMed

    Manns, M; Müller, M; Meyer zum Büschenfelde, K H

    1984-11-01

    Binding activity of antibodies against polymerized human serum albumin (pHSA) was measured in the serum of 348 patients with various hepatic and non-hepatic diseases and in the serum of 108 control persons. The methods used were passive hemagglutination (PH) with antigen loaded human erythrocytes and radial immunodiffusion (ID). In the PH-method only HBsAg-positive sera reacted. Blocking experiments with pHSA, polymerized bovine serum albumin (pBSA) and monomeric human serum albumin (mHSA) showed, that the PH-method measures HBsAG associated receptors for pHSA. In HBeAG-positive cases titers were significantly higher than in anti-HBe-positive sera. Using the ID-method it could be shown, that 40% of sera of patients with liver diseases (n = 272), 37% of patients with LED (n = 27), 72% of patients with rheumatoid arthritis (n = 32), 6% of patients with glomerulonephritis (n = 17) and 2% of normal persons (n = 108) reacted. These sera reacted in the immunodiffusion assay with pHSA and pBSA but not with mHSA. Autoantibodies against non species specific determinants of pHSA which are not specific for liver diseases and possibly due to disturbed immunoregulation can be demonstrated by immunodiffusion. They may possibly be modulators of the pHSA mediated binding of hepatitis B-virus to hepatocytes.

  6. Comparative studies on drug binding to the purified and pharmaceutical-grade human serum albumins: Bridging between basic research and clinical applications of albumin.

    PubMed

    Ashrafi-Kooshk, Mohammad Reza; Ebrahimi, Farangis; Ranjbar, Samira; Ghobadi, Sirous; Moradi, Nastaran; Khodarahmi, Reza

    2015-09-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, is a monomeric multidomain protein that possesses an extraordinary capacity for binding, so that serves as a circulating depot for endogenous and exogenous compounds. During the heat sterilization process, the structure of pharmaceutical-grade HSA may change and some of its activities may be lost. In this study, to provide deeper insight on this issue, we investigated drug-binding and some physicochemical properties of purified albumin (PA) and pharmaceutical-grade albumin (PGA) using two known drugs (indomethacin and ibuprofen). PGA displayed significantly lower drug binding capacity compared to PA. Analysis of the quenching and thermodynamic parameters indicated that intermolecular interactions between the drugs and the proteins are different from each other. Surface hydrophobicity as well as the stability of PGA decreased compared to PA, also surface hydrophobicity of PA and PGA increased upon drugs binding. Also, kinetic analysis of pseudo-esterase activities indicated that Km and Vmax parameters for PGA enzymatic activity are more and less than those of PA, respectively. This in vitro study demonstrates that the specific drug binding of PGA is significantly reduced. Such studies can act as connecting bridge between basic research discoveries and clinical applications.

  7. Curcumin-incorporated albumin nanoparticles and its tumor image

    NASA Astrophysics Data System (ADS)

    Gong, Guangming; Pan, Qinqin; Wang, Kaikai; Wu, Rongchun; Sun, Yong; Lu, Ying

    2015-01-01

    Albumin is an ideal carrier for hydrophobic drugs. This paper reports a facile route to develop human serum albumin (HSA)-curcumin (CCM) nanoparticles, in which β-mercaptoethanol (β-ME) acted as an inducer and CCM acted as a bridge. Fluorescence quenching and conformational changes in HSA-CCM nanoparticles occurred during assembly. Disulfide bonds and hydrophobic interactions may play a key role in assembly. HSA-CCM nanoparticles were about 130 nm in size, and the solubility of CCM increased by more than 500 times. The HSA-CCM nanoparticles could accumulate at the cytoplasm of tumor cells and target the tumor tissues. Therefore, HSA nanoparticles fabricated by β-ME denaturation are promising nanocarriers for hydrophobic substances from chemotherapy drugs to imaging probes.

  8. Electrochemical characterization of albumin protein on Ti-6AL-4V alloy immersed in a simulated plasma solution.

    PubMed

    Padilla, Norma; Bronson, Arturo

    2007-06-01

    The effect of oxygen and albumin on the electrochemical behavior of a Ti-6Al-4V alloy immersed in a simulated inorganic plasma (SIP) solution was studied with a rotating-cylindrical electrode configuration to focus on the surface/electrolyte reactions. Potentiokinetic scans and electrochemical impedance spectroscopy have been used to characterize the interface by determining the passive current density and capacitance. For the polarization scans, an albumin addition of 37.7 mg/cm(3) to the SIP solution (oxygenated and unoxygenated) decreased the passive current density, indicating a lowering of the corrosive rate. The surface capacitance for the Ti-6Al-4V alloy immersed in a SIP solution averaged 13 microF/cm(2), which transformed after albumin addition (37.7 mg/cm(3)) from a potential independent behavior to the capacitance ranging from 23 to 6 microF/cm(2) with increasing potentials from -800 to 1500 mV(SCE), respectively, indicative of albumin adsorption. Within the same potential range and albumin addition to oxygenated solutions, the capacitances expanded slightly with a similar decreasing trend from 31 to 6 microF/cm(2), although the capacitance depicts an interaction between the hydrated passive film and the adsorbed albumin from -550 to 500 mV(SCE) in which the capacitance plateaued at 15 microF/cm(2). The hydrated porous oxide film results from the porous rutile layer reacting with H(2)O(2) formed as an intermediary component of oxygen reduction at the Ti-6Al-4V surface. The passive film-albumin interaction would affect the processing of titanium alloys in their surface preparation for biocompatibility, as well as determining the reactivity of titanium alloys to proteins.

  9. Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

    PubMed

    Kang, Choong Mo; Kim, Hyunjung; Koo, Hyun-Jung; Park, Jin Won; An, Gwang Il; Choi, Joon Young; Lee, Kyung-Han; Kim, Byung-Tae; Choe, Yearn Seong

    2016-07-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates. PMID:27098932

  10. Differential sensing using proteins: exploiting the cross-reactivity of serum albumin to pattern individual terpenes and terpenes in perfume.

    PubMed

    Adams, Michelle M; Anslyn, Eric V

    2009-12-01

    There has been a growing interest in the use of differential sensing for analyte classification. In an effort to mimic the mammalian senses of taste and smell, which utilize protein-based receptors, we have introduced serum albumins as nonselective receptors for recognition of small hydrophobic molecules. Herein, we employ a sensing ensemble consisting of serum albumins, a hydrophobic fluorescent indicator (PRODAN), and a hydrophobic additive (deoxycholate) to detect terpenes. With the aid of linear discriminant analysis, we successfully applied our system to differentiate five terpenes. We then extended our terpene analysis and utilized our sensing ensemble for terpene discrimination within the complex mixtures found in perfume.

  11. Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes.

    PubMed

    Pantusa, Manuela; Stirpe, Andrea; Sportelli, Luigi; Bartucci, Rosa

    2010-05-01

    Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime. PMID:19350232

  12. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kasálková, Nikola Slepičková; Slepička, Petr; Kolská, Zdeňka; Hodačová, Petra; Kučková, Štěpánka; Švorčík, Václav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  13. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

    PubMed Central

    Yamamoto, N; Kumashiro, R; Yamamoto, M; Willett, N P; Lindsay, D D

    1993-01-01

    A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited. PMID:8225612

  14. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    PubMed Central

    2014-01-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly-l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly-l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation. PMID:24708858

  15. Interaction with Serum Albumin As a Factor of the Photodynamic Efficacy of Novel Bacteriopurpurinimide Derivatives

    PubMed Central

    Akimova, Akimova; Rychkov, G. N.; Grin, M. A.; Filippova, N. A.; Golovina, G. V.; Durandin, N. A.; Vinogradov, A. M.; Kokrashvili, T. A.; Mironov, A. F.; Shtil, A. A.; Kuzmin, V. A.

    2015-01-01

    Optimization of the chemical structure of antitumor photosensitizers (PSs) is aimed at increasing their affinity to a transport protein, albumin and irreversible light-induced tumor cell damage. Bacteriopurpurinimide derivatives are promising PSs thanks to their ability to absorb light in the near infrared spectral region. Using spectrophotometry, we show that two new bacteriopurpurinimide derivatives with different substituents at the N atoms of the imide exocycle and the pyrrole ring A are capable of forming non-covalent complexes with human serum albumin (HSA). The association constant (calculated with the Benesi-Hildebrand equation) for N-ethoxybacteriopurpurinimide ethyloxime (compound 1) is higher than that for the methyl ether of methoxybacteriopurpurinimide (compound 2) (1.18×105 M-1 vs. 1.26×104 M-1, respectively). Molecular modeling provides details of the atomic interactions between 1 and 2 and amino acid residues in the FA1 binding site of HSA. The ethoxy group stabilizes the position of 1 within this site due to hydrophobic interaction with the protein. The higher affinity of 1 for HSA makes this compound more potent than 2 in photodynamic therapy for cultured human colon carcinoma cells. Photoactivation of 1 and 2 in cells induces rapid (within a few minutes of irradiation) necrosis. This mechanism of cell death may be efficient for eliminating tumors resistant to other therapies. PMID:25927008

  16. Curcumin promotes fibril formation in F isomer of human serum albumin via amorphous aggregation.

    PubMed

    Mothi, Nivin; Muthu, Shivani A; Kale, Avinash; Ahmad, Basir

    2015-12-01

    We here describe the amyloid fibrils promoting behavior of curcumin, which ability to inhibit amyloid fibrillization of several globular proteins is well documented. Transmission electron microscopy (TEM), 90° light scattering (RLS), thioflavine T (ThT) and Congo red (CR) binding studies demonstrated that both F (pH3.4) and E (pH1.8) isomers of human serum albumin (HSA) in the absence and presence of curcumin initially converted into amorphous aggregates. Interestingly, only the sample containing F isomer preincubated with curcumin formed fibrils on incubation for longer period. We also found that curcumin strongly bind to the F isomer, alter its secondary, tertiary structures and thermal stability. We conclude that the conversion of intermediate states into amorphous aggregate to fibrils is dictated by its conformation. This study provides unique insights into ligand-controlled HSA aggregation pathway and should provide a useful model system to study both amorphous and the fibrillar aggregation of multidomain proteins.

  17. Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach.

    PubMed

    Pu, Hanlin; Jiang, Hua; Chen, Rongrong; Wang, Hongcui

    2014-08-01

    The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were -4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non-radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na(+), K(+), Li(+), Ni(2+), Ca(2+), Zn(2+) and Al(3+) were found to influence binding of the drug to protein. The 3D fluorescence, FT-IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA.

  18. Investigation the interaction of Daphnin with human serum albumin using optical spectroscopy and molecular modeling methods

    NASA Astrophysics Data System (ADS)

    Zhu, Jinhua; Wu, Liye; Zhang, Qingyou; Chen, Xingguo; Liu, Xiuhua

    2012-09-01

    The interaction between Daphnin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Daphnin can quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the protein secondary structure changed with increases of α-helices at the drug to protein molar ratio of 2. Furthermore, the thermodynamic functions ΔH0 and ΔS0 for the reaction were calculated to be 11.626 kJ mol-1 and 118.843 J mol-1 K-1 according to Van't Hoff equation. The thermodynamic parameters (ΔH0 and ΔS0) and the molecular modeling study indicated that hydrophobic force played an important role to stabilize the Daphnin-HSA complex, and Daphnin could bind within the subdomain IIA of the HSA.

  19. Novel self-assembly endows human serum albumin nanoparticles with an enhanced antitumor efficacy.

    PubMed

    Ding, Dawei; Tang, Xiaolei; Cao, Xiaoli; Wu, Jinhui; Yuan, Ahu; Qiao, Qian; Pan, Jing; Hu, Yiqiao

    2014-02-01

    Protein-based nanomedicine plays an important role in tumor chemotherapy due to their merits in bioavailability, biocompatibility, biodegradability, and low toxicity. In this study, we developed a novel method of preparing human serum albumin (HSA) nanoparticles for targeted delivery of paclitaxel (PTX) to tumors. HSA-PTX nanoparticles (NPs-PTX) were fabricated via unfolding of HSA in appropriate solution to expose more hydrophobic domains and consequent self-assembling into nanoparticles with added PTX. Via this self-assembly method, a desirable particle size (around 120 nm), a high drug loading (>20%), and a high encapsulation efficiency (near 100%) were obtained. PTX dispersed as an amorphous state in NPs-PTX and the secondary structures of HSA were maintained. In a cytotoxicity study, NPs-PTX displayed an enhanced cytotoxicity in MCF-7 and A549 cells. Confocal microscopy and flow cytometry revealed that the uptake of NPs-PTX was mediated by secreted protein acidic and rich in cysteine and "caveolar" transport. In H22 tumor-bearing mice, NPs-PTX displayed an increasing and everlasting tumor distribution, leading to slower tumor growth and longer mice survival than PTX. Therefore, this novel self-assembly method offers a much easier method to prepare PTX nanoparticles, provides better antitumor efficacy in vitro and in vivo, and more importantly, sets up a delivery platform for other hydrophobic drugs to improve their effectiveness in cancer therapy.

  20. Structural alterations of human serum albumin caused by glycative and oxidative stressors revealed by circular dichroism analysis.

    PubMed

    Monacelli, Fiammetta; Storace, Daniela; D'Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L; Pronzato, Maria A; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points. PMID:23702842

  1. Structural alterations of human serum albumin caused by glycative and oxidative stressors revealed by circular dichroism analysis.

    PubMed

    Monacelli, Fiammetta; Storace, Daniela; D'Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L; Pronzato, Maria A; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.

  2. Structural Alterations of Human Serum Albumin Caused by Glycative and Oxidative Stressors Revealed by Circular Dichroism Analysis

    PubMed Central

    Monacelli, Fiammetta; Storace, Daniela; D’Arrigo, Cristina; Sanguineti, Roberta; Borghi, Roberta; Pacini, Davide; Furfaro, Anna L.; Pronzato, Maria A.; Odetti, Patrizio; Traverso, Nicola

    2013-01-01

    The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points. PMID:23702842

  3. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins.

    PubMed

    Yadav, Indresh; Aswal, Vinod K; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins-cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  4. In vivo genome editing of the albumin locus as a platform for protein replacement therapy

    PubMed Central

    Sharma, Rajiv; Anguela, Xavier M.; Doyon, Yannick; Wechsler, Thomas; DeKelver, Russell C.; Sproul, Scott; Paschon, David E.; Miller, Jeffrey C.; Davidson, Robert J.; Shivak, David; Zhou, Shangzhen; Rieders, Julianne; Gregory, Philip D.; Holmes, Michael C.; Rebar, Edward J.

    2015-01-01

    Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) –mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases. PMID:26297739

  5. Interaction of Human Serum Albumin with Metal Protoporphyrins

    NASA Astrophysics Data System (ADS)

    Hu, Jie; Brancaleon, Lorenzo

    2015-03-01

    Fluorescence spectroscopy is widely used in biotechnology, nanotechnology, and molecular biophysics, since it can provide information on a wide range of molecular processes, e.g. the interactions of solvent molecules with fluorophores, conformational changes, and binding interactions etc. In this study, we present the photophysical properties of the interaction of human serum albumin (HSA) with a series of metal compound of Protoporphyrin IX (PPIX), including ZnPPIX, FePPIX, MgPPIX, MnPPIX and SnPPIX respectively, as well as the free base PPIX. Binding constants were retrieved independently using the Benesi-Hildebrand analysis of the porphyrin emission or absorption spectra and the fluorescence quenching (i.e. Stern-Volmer analysis) and reveal that the two methods yield a difference of approximately one order or magnitude between the two. Fluorescence lifetimes was used to probe whether binding of the porphyrin changes the conformation of the protein or if the interaction places the porphyrin at a location that can prompt resonance energy transfer with the lone Tryptophan residue. In recent years it has been discovered that HSA provides a specific binding site for metal-chelated protoporphyrins in subdomain IA. This has opened a novel field of study over the importance of this site for biomedical applications but it has also created the potential for a series of biotechnological applications of the HSA/protoporphyrin complexes. Our study provides a preliminary investigation of the interaction with metal-chelated protoporphyrins that had not been previously investigated.

  6. Probing the interaction of a new synthesized CdTe quantum dots with human serum albumin and bovine serum albumin by spectroscopic methods.

    PubMed

    Bardajee, Ghasem Rezanejade; Hooshyar, Zari

    2016-05-01

    A novel CdTe quantum dots (QDs) were prepared in aqueous phase via a facile method. At first, poly (acrylic amide) grafted onto sodium alginate (PAAm-g-SA) were successfully synthesized and then TGA capped CdTe QDs (CdTe-TGA QDs) were embed into it. The prepared CdTe-PAAm-g-SA QDs were optimized and characterized by transmission electron microscopy (TEM), thermo-gravimetric (TG) analysis, Fourier transform infrared (FT-IR), UV-vis and fluorescence spectroscopy. The characterization results indicated that CdTe-TGA QDs, with particles size of 2.90 nm, were uniformly dispersed on the chains of PAAm-g-SA biopolymer. CdTe-PAAm-g-SA QDs also exhibited excellent UV-vis absorption and high fluorescence intensity. To explore biological behavior of CdTe-PAAm-g-SA QDs, the interactions between CdTe-PAAm-g-SA QDs and human serum albumin (HSA) (or bovine serum albumin (BSA)) were investigated by cyclic voltammetry, FT-IR, UV-vis, and fluorescence spectroscopic. The results confirmed the formation of CdTe-PAAm-g-SA QDs-HSA (or BSA) complex with high binding affinities. The thermodynamic parameters (ΔG<0, ΔH<0 and ΔS<0) were indicated that binding reaction was spontaneous and van der Waals interactions and hydrogen-bond interactions played a major role in stabilizing the CdTe-PAAm-g-SA QDs-HSA (or BSA) complexes. The binding distance between CdTe-PAAm-g-SA QDs and HSA (or BSA)) was calculated about 1.37 nm and 1.27 nm, respectively, according to Forster non-radiative energy transfer theory (FRET). Analyzing FT-IR spectra showed that the formation of QDs-HSA and QDs-BSA complexes led to conformational changes of the HSA and BSA proteins. All these experimental results clarified the effective transportation and elimination of CdTe-PAAm-g-SA QDs in the body by binding to HSA and BSA, which could be a useful guideline for the estimation of QDs as a drug carrier.

  7. The conformation of serum albumin in solution: a combined phosphorescence depolarization-hydrodynamic modeling study.

    PubMed Central

    Ferrer, M L; Duchowicz, R; Carrasco, B; de la Torre, J G; Acuña, A U

    2001-01-01

    There is a striking disparity between the heart-shaped structure of human serum albumin (HSA) observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH. These two contrasting views could be reconciled if the protein were flexible enough to change its conformation in solution from that found in the crystal. To investigate this possibility we recorded the rotational motions in real time of an erythrosin-bovine serum albumin complex (Er-BSA) over an extended time range, using phosphorescence depolarization techniques. These measurements are consistent with the absence of independent motions of large protein segments in solution, in the time range from nanoseconds to fractions of milliseconds, and give a single rotational correlation time phi(BSA, 1 cP, 20 degrees C) = 40 +/- 2 ns. In addition, we report a detailed analysis of the protein hydrodynamics based on two bead-modeling methods. In the first, BSA was modeled as a triangular prismatic shell with optimized dimensions of 84 x 84 x 84 x 31.5 A, whereas in the second, the atomic-level structure of HSA obtained from crystallographic data was used to build a much more refined rough-shell model. In both cases, the predicted and experimental rotational diffusion rate and other hydrodynamic parameters were in good agreement. Therefore, the overall conformation in neutral solution of BSA, as of HSA, should be rigid, in the sense indicated above, and very similar to the heart-shaped structure observed in HSA crystals. PMID:11325741

  8. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  9. Comparative studies of the effects of copper sulfate and zinc sulfate on serum albumins

    NASA Astrophysics Data System (ADS)

    Plotnikova, O. A.; Melnikov, G. V.; Melnikov, A. G.; Kovalenko, A. V.

    2016-04-01

    The work is devoted to the study of the interaction of heavy metals with bovine serum albumin (BSA) and human serum albumin (HSA), by quenching of the intrinsic fluorescence of proteins and fluorescent probe pyrene by heavy metal ions. Sulfates of copper and zinc (CuSO4, ZnSO4) were taken as the metal salts. The value of the Stern-Volmer constants of quenching of intrinsic fluorescence of proteins and fluorescence probe pyrene reduced from Cu (II) to the Zn (II). It was experimentally found that the copper ions have a greater ability to fluorescence quenching, which is probably associated with the greater availability of protein chromophore groups to copper ions and with adsorbed fluorescent probe pyrene in the protein globule.

  10. Characterization and analytical application of Morin - Bovine serum albumin system by spectroscopic approaches

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Huang, Wei; Miao, Xiaowen; Tang, Bo

    2012-12-01

    It is found that the fluorescence intensity of Morin can be strongly quenched by proteins. Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the quenchment of Morin fluorescence was in proportion to the concentration of proteins in the range 0.0001-0.1000 g · L-1 for bovine serum albumin (BSA) and 0.0005-0.1000 g · L-1 for human serum albumin (HSA). The reaction mechanism indicates that proteins can bind with Morin at the 3-hydroxyl and the 4-carbonyl and form a non-fluorescence complex 4:1 molar ratio of Morin/BSA, which results in the fluorescence of Morin and BSA are all quenched.

  11. Evaluation of enantioselective binding of propanocaine to human serum albumin by ultrafiltration and electrokinetic chromatography under intermediate precision conditions.

    PubMed

    Martínez-Gómez, María Amparo; Escuder-Gilabert, Laura; Villanueva-Camañas, Rosa María; Sagrado, Salvador; Medina-Hernández, María José

    2012-03-15

    Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO-HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers' identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, logK(E1)=3.20±0.16 and log K(E2)=3.40±0.14, medium protein binding percentage, PB=48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES=K(E2)/K(E1)=1.5±0.3.

  12. Arginine and lysine reduce the high viscosity of serum albumin solutions for pharmaceutical injection.

    PubMed

    Inoue, Naoto; Takai, Eisuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2014-05-01

    Therapeutic protein solutions for subcutaneous injection must be very highly concentrated, which increases their viscosity through protein-protein interactions. However, maintaining a solution viscosity below 50 cP is important for the preparation and injection of therapeutic protein solutions. In this study, we examined the effect of various amino acids on the solution viscosity of very highly concentrated bovine serum albumin (BSA) and human serum albumin (HSA) at a physiological pH. Among the amino acids tested, l-arginine hydrochloride (ArgHCl) and l-lysine hydrochloride (LysHCl) (50-200 mM) successfully reduced the viscosity of both BSA and HSA solutions; guanidine hydrochloride (GdnHCl), NaCl, and other sodium salts were equally as effective, indicating the electrostatic shielding effect of these additives. Fourier transform infrared spectroscopy showed that BSA is in its native state even in the presence of ArgHCl, LysHCl, and NaCl at high protein concentrations. These results indicate that weakened protein-protein interactions play a key role in reducing solution viscosity. ArgHCl and LysHCl, which are also non-toxic compounds, will be used as additives to reduce the solution viscosity of concentrated therapeutic proteins.

  13. (Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

    PubMed Central

    Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

    2011-01-01

    In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

  14. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography

    PubMed Central

    Pfaunmiller, Erika L.; Hartmann, Mahli; Dupper, Courtney M.; Soman, Sony; Hage, David S.

    2012-01-01

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6–2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6 mm i.d. × 50 mm columns. These monoliths were also used to create 4.6 mm i.d. × 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5–6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  15. Electrokinetic characteristics of HSA dimer and its monolayers at mica.

    PubMed

    Kujda, Marta; Adamczyk, Zbigniew; Zapotoczny, Szczepan; Kowalska, Ewa

    2015-12-01

    Human serum albumin (HSA) dimer was synthesized in a reaction of the monomer with 1,6-bis(maleimido)hexane (BMH) cross-linker. Thorough physicochemical characteristics of the dimer were performed. They comprised the diffusion coefficient, hydrodynamic diameter, electrophoretic mobility as a function of pH, isoelectric point and electrokinetic charge. The adsorption of the dimer molecules at mica was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of adsorption was determined by a direct AFM imaging of single molecules over various substrate areas and interpreted in terms of the random sequential adsorption model. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow. It was also shown by these measurements that the desorption of the monomer under flow conditions was negligible. In this way, the amount of irreversibly bound dimer was quantitatively evaluated to be 0.5 mgm(-2) for ionic strength of 0.01 and pH 3.5 that is similar that to previous result obtained for the albumin monomer. This indicates that the dimer adsorption occurs mostly in the side-on orientation. Finally, the electrokinetic characteristics of the dimer monolayers on mica were performed by the streaming potential method. It was observed that for lower pHs the zeta potential of monolayers is much lower than the bulk zeta potential of the dimer molecules. This was attributed to a heterogeneous charge distribution. It was concluded that the well-characterized HSA dimer monolayers can be used for quantitatively determining ligand and drug binding that has an essential practical significance. PMID:26613862

  16. Electrokinetic characteristics of HSA dimer and its monolayers at mica.

    PubMed

    Kujda, Marta; Adamczyk, Zbigniew; Zapotoczny, Szczepan; Kowalska, Ewa

    2015-12-01

    Human serum albumin (HSA) dimer was synthesized in a reaction of the monomer with 1,6-bis(maleimido)hexane (BMH) cross-linker. Thorough physicochemical characteristics of the dimer were performed. They comprised the diffusion coefficient, hydrodynamic diameter, electrophoretic mobility as a function of pH, isoelectric point and electrokinetic charge. The adsorption of the dimer molecules at mica was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of adsorption was determined by a direct AFM imaging of single molecules over various substrate areas and interpreted in terms of the random sequential adsorption model. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow. It was also shown by these measurements that the desorption of the monomer under flow conditions was negligible. In this way, the amount of irreversibly bound dimer was quantitatively evaluated to be 0.5 mgm(-2) for ionic strength of 0.01 and pH 3.5 that is similar that to previous result obtained for the albumin monomer. This indicates that the dimer adsorption occurs mostly in the side-on orientation. Finally, the electrokinetic characteristics of the dimer monolayers on mica were performed by the streaming potential method. It was observed that for lower pHs the zeta potential of monolayers is much lower than the bulk zeta potential of the dimer molecules. This was attributed to a heterogeneous charge distribution. It was concluded that the well-characterized HSA dimer monolayers can be used for quantitatively determining ligand and drug binding that has an essential practical significance.

  17. Evidence of Energy Transfer from Tryptophan to BSA/HSA Protected Gold Nanoclusters

    PubMed Central

    Raut, Sangram; Chib, Rahul; Butler, Susan; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2015-01-01

    This work reports on the chromophores interactions within protein-protected gold nanoclusters. We conducted spectroscopic studies of fluorescence emissions originated from gold nanoclusters and intrinsic tryptophan (Trp) in BSA or HSA proteins. Both, steady state fluorescence and lifetime measurements show a significant Forster resonance energy transfer (FRET) from Trp to the gold nanocluster. Tryptophan lifetimes in the case of protein-protected gold nanoclusters are 2.6ns and 2.3ns for BSA and HSA Au clusters while 5.8ns for native BSA and 5.6 for native HSA. The apparent distances from Trp to gold nanocluster emission center, we estimated as 24.75A0 for BSA and 23.80A0 for HSA. We also studied a potassium iodide (KI) quenching of protein-protected gold nanoclusters and compared with the quenching of BSA and HAS alone. The rates of Trp quenching were smaller in BSA-Au and HSA-Au nanoclusters than in the case of free proteins, which is consistent with shorter lifetime of quenched Trp(s) and lower accessibility for KI. While Trp residues were quenched by KI, the emissions originated from nanoclusters were practically unquenched. In summary, for BSA and HSA Au clusters, we found 55% and 59% energy transfer efficiency respectively from tryoptophan to gold clusters. We believe this interaction can be used to our advantage in terms of developing resonance energy transfer based sensing applications. PMID:26767113

  18. Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin.

    PubMed

    Paul, Bijan K; Ghosh, Narayani; Mukherjee, Saptarshi

    2015-10-15

    Herein, the binding interaction of a potential chemotherapeutic antibacterial drug norfloxacin (NOF) with a serum transport protein, human serum albumin (HSA), is investigated. The prototropic transformation of the drug (NOF) is found to be remarkably modified following interaction with the protein as manifested through significant modulations of the photophysics of the drug. The predominant zwitterionic form of NOF in aqueous buffer phase undergoes transformation to the cationic form within the protein-encapsulated state. This implies the possible role of electrostatic interaction force in NOF-HSA binding. This postulate is further substantiated from the effect of ionic strength on the interaction process. To this end, the detailed study of the thermodynamics of the drug-protein interaction process from isothermal titration calorimetric (ITC) experiments is found to unfold the signature of electrostatic as well as hydrophobic interaction forces underlying the binding process. Thus, interplay of more than one interaction forces is argued to be responsible for the overall drug-protein binding. The ITC results reveal an important finding in terms of enthalpy-entropy compensation (EEC) characterizing the NOF-HSA binding. The effect of drug-binding on the native protein conformation has also been evaluated from circular dichroism (CD) spectroscopy which unveils partial rupture of the protein secondary structure. In conjunction to this, the functionality of the native protein (in terms of esterase-like activity) is found to be lowered as a result of binding with NOF. The AutoDock-based docking simulation unravels the probable binding location of NOF within the hydrophilic subdomain IA of HSA. The present program also focuses on exploring the dynamical aspects of the drug-protein interaction scenario. The rotational-relaxation dynamics of the protein-bound drug reveals the not-so-common "dip-and-rise" pattern.

  19. Ratio of C-Reactive Protein to Albumin Predicts Muscle Mass in Adult Patients Undergoing Hemodialysis

    PubMed Central

    Chen, Yu-Tong; Wu, Pei-Yu; Chen, Hsi-Hsien; Chen, Tso-Hsiao; Hsu, Yung-Ho

    2016-01-01

    Recent studies have indicated that the ratio of C-reactive protein to albumin (CRP–Alb ratio) is associated with clinical outcomes in patients with disease. We examined the predictive value of this ratio in patients undergoing hemodialysis (HD). In this cross-sectional study, 91 eligible adult HD patients were analyzed, and the correlation between the CRP–Alb ratio and skeletal muscle mass normalized for body weight (SMM/wt; estimated using a bioelectrical impedance analyzer) was investigated. The mean age of the study participants was 54.9 ± 6.6 years (ranging from 27 to 64 years); 43 (47.2%) were men. The mean values for the SMM/wt were 39.1% ± 5.4%. The CRP–Alb ratio was found to be negatively correlated with SMM/wt (r = −0.33, P = 0.002) and creatinine (r = −0.20, P = 0.056). All the univariate significant and nonsignificant relevant covariates were selected for multivariable stepwise regression analysis. We determined that the homeostasis model assessment-estimated insulin resistance and CRP–Alb ratio were independent risk determinants for SMM/wt (βHOMA-IR = −0.18 and βCRP–Alb ratio = −3.84, adjusted R2 = 0.32). This study indicated that the CRP–Alb ratio may help clinicians in predicting muscle mass in adult patients undergoing HD. PMID:27768746

  20. Impact of protein modification on the protein corona on nanoparticles and nanoparticle-cell interactions.

    PubMed

    Treuel, Lennart; Brandholt, Stefan; Maffre, Pauline; Wiegele, Sarah; Shang, Li; Nienhaus, G Ulrich

    2014-01-28

    Recent studies have firmly established that cellular uptake of nanoparticles is strongly affected by the presence and the physicochemical properties of a protein adsorption layer around these nanoparticles. Here, we have modified human serum albumin (HSA), a serum protein often used in model studies of protein adsorption onto nanoparticles, to alter its surface charge distribution and investigated the consequences for protein corona formation around small (radius ∼5 nm), dihydrolipoic acid-coated quantum dots (DHLA-QDs) by using fluorescence correlation spectroscopy. HSA modified by succinic anhydride (HSAsuc) to generate additional carboxyl groups on the protein surface showed a 3-fold decreased binding affinity toward the nanoparticles. A 1000-fold enhanced affinity was observed for HSA modified by ethylenediamine (HSAam) to increase the number of amino functions on the protein surface. Remarkably, HSAsuc formed a much thicker protein adsorption layer (8.1 nm) than native HSA (3.3 nm), indicating that it binds in a distinctly different orientation on the nanoparticle, whereas the HSAam corona (4.6 nm) is only slightly thicker. Notably, protein binding to DHLA-QDs was found to be entirely reversible, independent of the modification. We have also measured the extent and kinetics of internalization of these nanoparticles without and with adsorbed native and modified HSA by HeLa cells. Pronounced variations were observed, indicating that even small physicochemical changes of the protein corona may affect biological responses.

  1. Spectroscopy and Molecular Modeling Study on Binding of Nickel Phthalocyanine to Human Serum Albumin.

    PubMed

    Dezhampanah, Hamid; Firouzi, Roghaye; Hasani, Leila

    2016-01-01

    The interaction of nickel tetra sulfunated phthalocyanine( NiTSPc) with human serum albumin (HSA), in 20 mM phosphate buffer pH 7.4 was investigated using advanced techniques including fluorescence, synchronous fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopy and molecular docking. The fluorescence quenching measurements showed a single binding site on HSA for NiTSPc with the binding constant (Kb) value equals to 1.26×106 at 25°C. The results showed that quenching mechanism of HSA by NiTSPc was of dynamic type. The results from FTIR and CD spectroscopies demonstrated that NiTSPc binds to amino acid residues of the main polypeptide chain in protein destroying the hydrogen bonding network. The corresponding thermodynamic parameters were then calculated by analysis of fluorescence data using van't Hoff plot. These data indicated that driving force for interaction was mainly hydrophobic in nature and the process was entropy driven. The information obtained from CD, FT-IR and synchronous fluorescence spectroscopies revealed that both microenvironment and conformation of HSA was changed. Molecular docking study confirmed the binding mode obtained by experimental data. PMID:27449940

  2. Computational studies of the binding mechanisms of fullerenes to human serum albumin.

    PubMed

    Li, Jinyu; Jiang, Lizhi; Zhu, Xiaolei

    2015-07-01

    Fullerene and its derivatives show promising prospects for applications in a vast array of biological systems. A key aspect concerning their biomedical applications is how they interact with proteins from molecular levels, which is still poorly understood. In the current study, we investigated the structural and thermodynamic basis of the interactions between two pharmacologically relevant fullerene derivatives and human serum albumin (HSA) using molecular docking, molecular dynamics simulations, and binding free energy calculations. Our results demonstrate that fullerenes steadily bind with HSA at the interfacial cavity formed by subdomains IIA and IIIA. In agreement with available experimental data, our simulations show that the global structure of HSA becomes more compact in the presence of fullerene, while local structural dynamics of the binding cavity behaves diversely depending on the chemical properties of bound fullerenes. Binding free energy calculations confirmed that the interactions between fullerenes and HSA are dominantly stabilized by van der Waals forces and they further allowed the identification of key residues involved in fullerene binding. The structural and energetic insights obtained from this work may help for the development of fullerene-based drug delivery devices and therapeutic agents with improved biological profile.

  3. Characterization of the interaction between 8-bromoadenosine with human serum albumin and its analytical application

    NASA Astrophysics Data System (ADS)

    Cui, Fengling; Yan, Yinghua; Zhang, Qiangzhai; Yao, Xiaojun; Qu, Guirong; Lu, Yan

    2009-11-01

    This study was designed to examine the interaction of 8-bromoadenosine with human serum albumin (HSA) by fluorescence spectroscopy in combination with molecular modeling under simulative physiological conditions. The results of fluorescence measurements indicate that 8-bromoadenosine has a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. The binding constants ( K) at different temperatures and thermodynamic parameters, enthalpy changes (Δ H) and entropy changes (Δ S) were calculated according to the fluorescence data. The results showed that the hydrophobic force played the major role in the binding of 8-bromoadenosine to HSA. The fluorescence experimental results were in agreement with the results obtained by molecular modeling study. The effects of some normal positive and negative ions on the binding constants were also discussed. Moreover, the synchronous fluorescence technique was used to characterize the interaction of 8-bromoadenosine to HSA and successfully applied to determine the total proteins in human serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.

  4. Interaction of prometryn to human serum albumin: insights from spectroscopic and molecular docking studies.

    PubMed

    Wang, Yaping; Zhang, Guowen; Wang, Langhong

    2014-01-01

    Prometryn possesses much potential hazard to environment because of its chemical stability and biological toxicity. Here, the binding properties of prometryn with human serum albumin (HSA) and the protein structural changes were determined under simulative physiological conditions (pH 7.4) by multispectroscopic methods including fluorescence, UV-vis absorption, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, coupled with molecular modeling technique. The result of fluorescence titration suggested that the fluorescence quenching of HSA by prometryn was considered as a static quenching procedure. The negative enthalpy change (ΔH(○)) and positive entropy change (ΔS(○)) values indicated that the binding process was governed mainly by hydrophobic interactions and hydrogen bonds. The site marker displacement experiments suggested the location of prometryn binding to HSA was Sudlow's site I in subdomain IIA. Furthermore, molecular docking studies revealed prometryn can bind in the large hydrophobic activity of subdomain IIA. Analysis of UV-vis absorption, synchronous fluorescence, CD and FT-IR spectra demonstrated that the addition of prometryn resulted in rearrangement and conformational alteration of HSA with reduction in α-helix and increases in β-sheet, β-turn and random coil structures. This work provided reasonable model helping us further understand the transportation, distribution and toxicity effect of prometryn when it spreads into human blood serum. PMID:24485317

  5. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  6. Reply to commentary on "Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole"

    NASA Astrophysics Data System (ADS)

    Punith, Reeta; Seetharamappa, J.

    2015-03-01

    The study by Punith and Seetharamappa [1] presents the effect of an anticancer drug, anastrozole (AZ), on the secondary structure of human serum albumin (HSA) and bovine serum albumin (BSA). Spectroscopic methods used in the study are absorption, CD, FTIR, synchronous and 3D fluorescence. The study reports that one molecule of AZ spontaneously bind to the protein on one site with hydrophobic interactions. As a result, almost 30% of the secondary structure of HSA is altered. This last conclusion is based on the results from CD and FTIR spectroscopy. Although this commentary is mainly intended to point out the misuse of FTIR data, there are also questions raised regarding other sections of the study.

  7. Quantitative parameters of complexes of tris(1-alkylindol-3-yl)methylium salts with serum albumin: Relevance for the design of drug candidates.

    PubMed

    Durandin, Nikita A; Tsvetkov, Vladimir B; Bykov, Evgeny E; Kaluzhny, Dmitry N; Lavrenov, Sergey N; Tevyashova, Anna N; Preobrazhenskaya, Maria N

    2016-09-01

    Triarylmethane derivatives are extensively investigated as antitumor and antibacterial drug candidates alone and as photoactivatable compounds. In the series of tris(1-alkylindol-3-yl)methylium salts (TIMs) these two activities differed depending on the length of N-alkyl chain, with C4-5 derivatives being the most potent compared to the shorter or longer chain analogs and to the natural compound turbomycin A (no N-substituent). Given that the human serum albumin (HSA) is a major transporter protein with which TIMs can form stable complexes, and that the formation of these complexes might be advantageous for phototoxicity of TIMs we determined the quantitative parameters of TIMs-HSA binding using spectroscopic methods and molecular docking. TIMs bound to HSA (1:1 stoichiometry) altered the protein's secondary structure by changing the α-helix/β-turn ratio. The IIa subdomain (Sudlow site I) is the preferred TIM binding site in HSA as determined in competition experiments with reference drugs ibuprofen and warfarin. The values of binding constants increased with the number of CH2 groups from 0 to 6 and then dropped down for C10 compound, a dependence similar to the one observed for cytocidal potency of TIMs. We tend to attribute this non-linear dependence to an interplay between hydrophobicity and steric hindrance, the two key characteristics of TIMs-HSA complexes calculated in the molecular docking procedure. These structure-activity relationships provide evidence for rational design of TIMs-based antitumor and antimicrobial drugs. PMID:27475780

  8. Radiochemical analysis of Tc-99m human serum albumin with high-pressure liquid chromatography: concise communication.

    PubMed

    Vallabhajosula, S; Goldsmith, S J; Pollina, R; Lipszyc, H

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT = 17 min) and pertechnetate (RT = 28.5 min). When analysis was performed on Tc-99m HSA preparations, Tc-99m radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining Tc-99m was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a Tc-99m HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  9. Radiochemical analysis of Tc-99m human serum albumin with high-pressure liquid chromatography: concise communication

    SciTech Connect

    Vallabhajosula, S.; Goldsmith, S.J.; Pollina, R.; Lipszyc, H.

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT = 17 min) and pertechnetate (RT = 28.5 min). When analysis was performed on Tc-99m HSA preparations, Tc-99m radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining Tc-99m was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a Tc-99m HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  10. Radiochemical analysis of /sup 99m/Tc human serum albumin with high-pressure liquid chromatography: concise communication

    SciTech Connect

    Vallabhajosula, S.; Goldsmith, S.J.; Pollina, R.; Lipszyc, H.

    1982-04-01

    High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT . 17 min) and pertechnetate (RT . 28.5 min). When analysis was performed on /sup 99m/Tc HSA preparations, /sup 99m/Tc radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining /sup 99m/Tc was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a /sup 99m/Tc HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.

  11. 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

  12. Structural studies on serum albumins under green light irradiation.

    PubMed

    Comorosan, Sorin; Polosan, Silviu; Popescu, Irinel; Ionescu, Elena; Mitrica, Radu; Cristache, Ligia; State, Alina Elena

    2010-10-01

    This paper presents two new experimental results: the protective effect of green light (GL) on ultraviolet (UV) denaturation of proteins, and the effect of GL on protein macromolecular structures. The protective effect of GL was revealed on two serum albumins, bovine (BSA) and human (HSA), and recorded by electrophoresis, absorption, and circular dichroism spectra. The effect of GL irradiation on protein structure was recorded by using fluorescence spectroscopy and electrophoresis. These new effects were modeled by quantum-chemistry computation using Gaussian 03 W, leading to good fit between theoretical and experimental absorption and circular dichroism spectra. A mechanism for these phenomena is suggested, based on a double-photon absorption process. This nonlinear effect may lead to generation of long-lived Rydberg macromolecular systems, capable of long-range interactions. These newly suggested systems, with macroscopic quantum coherence behaviors, may block the UV denaturation processes. PMID:20473754

  13. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumin on the Synthesis and Stability of Gold Nanostructures

    NASA Astrophysics Data System (ADS)

    Miranda, Érica; Tofanello, Aryane; Brito, Adrianne; Lopes, David; Giacomelli, Fernando; Albuquerque, Lindomar; Costa, Fanny; Ferreira, Fabio; Araujo-Chaves, Juliana; de Castro, Carlos; Nantes, Iseli

    2016-03-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from fifteen days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After two months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination

  14. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    NASA Astrophysics Data System (ADS)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  15. Effect of CdTe quantum dots size on the conformational changes of human serum albumin: results of spectroscopy and isothermal titration calorimetry.

    PubMed

    Yang, Bingjun; Liu, Rutao; Hao, Xiaopeng; Wu, Yongzhong; Du, Jie

    2013-10-01

    Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)-cadmium telluride (CdTe) QDs was added, and GSH-CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH-CdTe QDs was formed, the UV-vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH-CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.

  16. The Five-To-Six-Coordination Transition of Ferric Human Serum Heme-Albumin Is Allosterically-Modulated by Ibuprofen and Warfarin: A Combined XAS and MD Study

    PubMed Central

    Bionducci, Monica; Fanali, Gabriella; Meli, Massimiliano; Colombo, Giorgio; Fasano, Mauro; Ascenzi, Paolo; Mobilio, Settimio

    2014-01-01

    Human serum albumin (HSA) is involved physiologically in heme scavenging; in turn, heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, the allosteric effect of ibuprofen and warfarin on the local atomic structure around the ferric heme-Fe (heme-Fe(III)) atom of HSA-heme-Fe (HSA-heme-Fe(III)) has been probed by Fe-K edge X-ray absorption spectroscopy (XAS). The quantitative analysis of the Fe-K edge extended X-ray absorption fine structure (EXAFS) signals and modeling of the near edge (XANES) spectral features demonstrated that warfarin and ibuprofen binding modify the local structure of the heme-Fe(III). Combined XAS data analysis and targeted molecular dynamics (MD) simulations provided atomic resolution insights of protein structural rearrangements required to accommodate the heme-Fe(III) upon ibuprofen and warfarin binding. In the absence of drugs, the heme-Fe(III) atom is penta-coordinated having distorted 4+1 configuration made by the nitrogen atoms of the porphyrin ring and the oxygen phenoxy atom of the Tyr161 residue. MD simulations show that ibuprofen and warfarin association to the secondary fatty acid (FA) binding site 2 (FA2) induces a reorientation of domain I of HSA-heme-Fe(III), this leads to the redirection of the His146 residue providing an additional bond to the heme-Fe(III) atom, providing the 5+1 configuration. The comparison of Fe-K edge XANES spectra calculated using MD structures with those obtained experimentally confirms the reliability of the proposed structural model. As a whole, combining XAS and MD simulations it has been possible to provide a reliable model of the heme-Fe(III) atom coordination state and to understand the complex allosteric transition occurring in HSA-heme-Fe(III) upon ibuprofen and warfarin binding. PMID:25153171

  17. Chemical exchange saturation transfer (CEST) properties of albumin-binding and gold nanoparticle-bound Eu (III) chelates

    NASA Astrophysics Data System (ADS)

    Melendez, Milleo Dalmacio

    CEST agents derived from paramagnetic complexes, commonly referred to as PARACEST agents represent a new class of MRI contrast agents that respond to biological information such as pH, temperature, redox, and metabolite levels. In this work, CEST properties of two novel PARACEST agents were investigated upon binding to human serum albumin (HSA) and conjugation to gold nanoparticles (Au NPs). CEST properties of [EuDOTA(O-Et-Tyr)4] -when bound to HSA was studied to address the effect of proteins on CEST. The interaction of this Eu3+ complex to HSA was investigated by performing fluorescent probe displacement studies and it was found to bind HSA at two different binding pockets, the normal warfarin and dansyl glycine binding sites. The lipophilic pendant arms and the negative charge of this complex contribute to the favorable protein binding. However, the CEST signal was reduced 2-fold upon binding to HSA. The exchangeable protons on HSA provide a large proton pool that can exchange with the bound H 2O, competing for the exchange between bulk and bound water. Au NPs coated with [EuDOTA(CAM)4]3+ were prepared using the Brust method and characterized by measuring the CEST properties. The water residence lifetime for the Au-Eu NP conjugates increased 2-fold compared to the free Eu3+ complex presumably, as a result of the formation of hindered structure between the particle and the Eu3+ complex. Sensitivity enhancement in CEST was achieved by slowing down water exchange and increasing the number of exchangeable groups on the agent using Au-Eu NP conjugates. The CEST properties of small molecule PARACEST agents were shown to vary depending on the platform used in assembling larger adducts.

  18. Molecular modeling and spectroscopic studies on binding of 2,6-bis[4-(4-amino-2-trifluoromethylphenoxy)benzoyl] pyridine to human serum albumin

    NASA Astrophysics Data System (ADS)

    He, Wen-ying; Chen, Hui-juan; Sheng, Fen-ling; Yao, Xiao-jun

    2009-10-01

    BAFP (2,6-bis[4-(4-amino-2-trifluoromethylphenoxy)benzoyl] pyridine), a synthesized polyimide compound, was exploited for the first time to analyze its interaction with human serum albumin (HSA) by molecular modeling, fluorescence and Fourier transform infrared attenuated total reflection spectroscopy (FTIR ATR) with drug concentrations of 3.3 × 10 -6 to 3.0 × 10 -5 mol L -1. Molecular docking was performed to reveal the possible binding mode. The results suggested that BAFP can strongly bind to human serum albumin (HSA) and the primary binding site of BAFP is located in site II of HSA, which is supported by the results from the competitive experiment. The binding constants for the interaction of BAFP with HSA have been evaluated from relevant fluorescence data at different temperatures (296, 303, 310 and 308 K). The alterations of the protein secondary structure in the presence of BAFP in aqueous solution were quantitatively calculated by the evidences from FTIR ATR spectroscopes. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction, which is also in good agreement with the results of molecule modeling study. The enthalpy change Δ H0, the free energy change Δ G0 and the entropy change Δ S0 of 296 K were calculated to be -7.75, -27.68 kJ mol -1 and 67.33 J mol -1 K -1, respectively.

  19. Grading of severity of the condition in burn patients by serum protein and albumin/globulin studies.

    PubMed

    Kumar, Pramod

    2010-07-01

    Capillary permeability increases after inflammation with consequent leak of fluid, electrolytes, and proteins. The albumin molecule size being smaller (69 kDa) than the globulin molecule (90-156 kDa) will leak relatively at an early stage of the disease (with moderate increase in capillary pore size) than globulin leading to albumin/globulin reversal. In cases with severe permeability changes with rapid progression to larger pore size with simultaneous leak of both albumin and globulin, albumin/globulin reversal will not occur. In this study estimation the serum protein and albumin/globulin (A/G) ratio at frequent intervals was done to grade the severity of the condition of burn patients by assessing the severity of capillary leak.A total of 61 admitted patients (from March 2002 to December 2004) based on the protein values were divided into 3 groups (group 1: 6-8 g/dL, group 2: 5.1-5.9 g/dL, group 3: < or =5.0 g/dL), and all the patients who showed change in their protein levels during the study were shifted to appropriate group and were classified as group shifters. The mean survival time and mortality of various groups were compared, and A/G ratio of all the expired cases was analyzed.Group 3 patients showed higher mortality (95%) as compared to that in other groups (group 1 and 2: 0% each and group shifters: 30.2%). Median survival time of group 3 was significantly low as compared to that of group 1 (P < 0.0026), group 2 (P < 0.0006), and group shifters (P < 0.0000). In group shifters the mean time (days) required for shifting from one group to other just before death or discharge in survivors was significantly higher than that in expired cases. Of 26 cases expired during the study, initial A/G ratio at the time of first assigning the group was not reversed in 22 cases (84.6%).The study concluded that the severity (indicated by lower serum protein values) and speed (judged by A/G ratio changes and median survival time analysis) of capillary permeability changes

  20. Glutaraldehyde mediated conjugation of amino-coated magnetic nanoparticles with albumin protein for nanothermotherapy.

    PubMed

    Zhao, Lingyun; Yang, Bing; Dai, Xiaochen; Wang, Xiaowen; Gao, Fuping; Zhang, Xiaodong; Tang, Jintian

    2010-11-01

    A novel bioconjugation of amino saline capped Fe3O4 magnetic nanoparticles (MNPs) with bovine serum albumin (BSA) was developed by applying glutaraldehyde as activator. Briefly, Fe3O4 MNs were synthesized by the chemical co-precipitation method. Surface modification of the prepared MNPs was performed by employing amino saline as the coating agent. Glutaraldehyde was further applied as an activation agent through which BSA was conjugated to the amino-coated MNPs. The structure of the BSA-MNs was confirmed by FTIR analysis. Physico-chemical characterizations of the BSA-MNPs, such as surface morphology, surface charge and magnetic properties were investigated by Transmission Electron Microscopy (TEM), zeta-Potential and Vibrating Sample Magnetometer (VSM), etc. Magnetic inductive heating characteristics of the BSA-MNPs were analyzed by exposing the MNPs suspension (magnetic fluid) under alternative magnetic field (AMF). The results demonstrate that BSA was successfully conjugated with amino-coated MNs mediated through glutaraldehyde activation. The nanoparticles were spherical shaped with approximately 10 nm diameter. Possessing ideal magnetic inductive heating characteristics, which can generate very rapid and efficient heating while upon AMF exposure, BSA-MNPs can be applied as a novel candidature for magnetic nanothermotherapy for cancer treatment. In vitro cytotoxicity study on the human hepatocellular liver carcinoma cells (HepG-2) indicates that BSA-MNP is an efficient agent for cancer nanothermotherapy with satisfied biocompatibility, as rare cytotoxicity was observed in the absence of AMF. Moreover, our investigation provides a methodology for fabrication protein conjugated MNPs, for instance monoclonal antibody conjugated MNPs for targeting cancer nanothermotherapy. PMID:21137877

  1. Forster resonance energy transfer in the system of human serum albumin-xanthene dyes

    NASA Astrophysics Data System (ADS)

    Kochubey, V. I.; Pravdin, A. B.; Melnikov, A. G.; Konstantinova, I.; Alonova, I. V.

    2016-04-01

    The processes of interaction of fluorescent probes: eosin and erythrosine with human serum albumin (HSA) were studied by the methods of absorption and fluorescence spectroscopy. Extinction coefficients of probes were determined. Critical transfer radius and the energy transfer efficiency were defined by fluorescence quenching of HSA. Analysis of the excitation spectra of HSA revealed that the energy transfer process is carried out mainly between tryptophanyl and probes.

  2. Simultaneous photometric determination of albumin and total protein in animal blood plasma employing a multicommutated flow system to carried out on line dilution and reagents solutions handling

    NASA Astrophysics Data System (ADS)

    Luca, Gilmara C.; Reis, Boaventura F.

    2004-02-01

    An automatic flow procedure for the simultaneous determination of albumin and total protein in blood plasma samples is proposed. The flow network comprised a set of three-way solenoid valves assembled to implement the multicommutation. The flow set up was controlled by means of a computer equipped with an electronic interface card which running a software wrote in QUICKBASIC 4.5 performed on line programmed dilution to allow the determination of both albumin and total protein in blood plasma. The photometric methods based on Bromocresol Green and Biuret reagents were selected for determination of albumin and total protein, respectively. Two LEDs based photometers coupled together the flow cells were employed as detector. After the adjustment of the operational parameters the proposed system presented the following features: an analytical throughput of 45 sample processing per hour for two analytes; relative standard deviations of 1.5 and 0.8% ( n=10) for a typical sample presenting 34 g l -1 albumin and 90 g l -1 total protein, respectively; linear responses ranging from 0 to 15 g l -1 albumin ( r=0.998) and total protein ( r=0.999); sample and reagents consumption, 140 μl serum solution, 0.015 mg VBC and 0.432 mg CuSO 4 per determination, respectively. Applying the paired t-test between results obtained using the proposed system and reference methods no significant difference at 95 and 90% confidence level for albumin and total protein, respectively, were observed.

  3. Simultaneous photometric determination of albumin and total protein in animal blood plasma employing a multicommutated flow system to carried out on line dilution and reagents solutions handling.

    PubMed

    Luca, Gilmara C; Reis, Boaventura F

    2004-02-01

    An automatic flow procedure for the simultaneous determination of albumin and total protein in blood plasma samples is proposed. The flow network comprised a set of three-way solenoid valves assembled to implement the multicommutation. The flow set up was controlled by means of a computer equipped with an electronic interface card which running a software wrote in QUICKBASIC 4.5 performed on line programmed dilution to allow the determination of both albumin and total protein in blood plasma. The photometric methods based on Bromocresol Green and Biuret reagents were selected for determination of albumin and total protein, respectively. Two LEDs based photometers coupled together the flow cells were employed as detector. After the adjustment of the operational parameters the proposed system presented the following features: an analytical throughput of 45 sample processing per hour for two analytes; relative standard deviations of 1.5 and 0.8% (n=10) for a typical sample presenting 34 g l(-1) albumin and 90 g l(-1) total protein, respectively; linear responses ranging from 0 to 15 g l(-1) albumin (r=0.998) and total protein (r=0.999); sample and reagents consumption, 140 microl serum solution, 0.015 mg VBC and 0.432 mg CuSO4 per determination, respectively. Applying the paired t-test between results obtained using the proposed system and reference methods no significant difference at 95 and 90% confidence level for albumin and total protein, respectively, were observed.

  4. On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

    NASA Astrophysics Data System (ADS)

    Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2010-09-01

    The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

  5. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    PubMed Central

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  6. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    PubMed

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  7. The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins.

    PubMed

    Duff, Michael R; Kumar, Challa V

    2009-11-01

    Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.

  8. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

    PubMed

    Zhang, Guowen; Ma, Yadi

    2013-01-15

    The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA.

  9. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

    PubMed

    Zhang, Guowen; Ma, Yadi

    2013-01-15

    The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA. PMID:23122082

  10. Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Szkudlarek, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.; Sułkowska, A.

    2016-01-01

    The modification of human serum albumin (HSA) structure by non-enzymatic glycation is one of the underlying factors that contribute to the development of complications of diabetes and neurodegenerative diseases. The aim of the present work was to estimate how glycation of HSA altered its tertiary structure. Changes of albumin conformation were investigated by comparison of glycated (gHSA) and non-glycated human serum albumin (HSA) absorption spectra, red edge excitation shift (REES) and synchronous spectra. Effect of glycation on human serum albumin tertiary structure was also investigated by 1H NMR spectroscopy. Formation of gHSA Advanced Glycation End-products (AGEs) caused absorption of UV-VIS light between 310 nm and 400 nm while for non-glycated HSA in this region no absorbance has been registered. Analysis of red edge excitation shift effect allowed for observation of structural changes of gHSA in the hydrophobic pocket containing the tryptophanyl residue. Moreover changes in the microenvironment of tryptophanyl and tyrosyl residues brought about AGEs on the basis of synchronous fluorescence spectroscopy have been confirmed. The influence of glycation process on serum albumin binding to 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS), has been studied. Fluorescence analysis showed that environment of both binding site I and II is modified by galactose glycation.

  11. Magnetic molecularly imprinted polydopamine nanolayer on multiwalled carbon nanotubes surface for protein capture.

    PubMed

    Yin, Yuli; Yan, Liang; Zhang, Zhaohui; Wang, Jing

    2015-11-01

    A novel, facile and low cost process for imprinting protein on the surface of magnetic multiwalled carbon nanotubes (MMWNTs) was developed using human serum albumin (HSA) as the template and dopamine as the functional monomer. The magnetic imprinted polymers were characterized with transmission electron microscope (TEM), scanning electron microscope (SEM), Fourier-transform infrared spectrometry (FT-IR), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA) in detail. The maximum adsorption capacity of the magnetic imprinted polymers toward HSA was 66.23 mg g(-1) and it took 20 min to achieve the adsorption equilibrium. The magnetic imprinted polymers exhibited the specific selective adsorption toward HSA. Coupled with high performance liquid chromatography (HPLC) analysis, the magnetic imprinted polymers were used to solid-phase extract and detect HSA in urine samples successfully with the recoveries of 91.95-97.8%.

  12. Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications.

    PubMed

    Santhosh, Mallesh; Chinnadayyala, Somasekhar R; Singh, Naveen K; Goswami, Pranab

    2016-10-01

    Human serum albumin (HSA)-stabilized Au18 nanoclusters (AuNCs) were synthesized and chemically immobilized on an Indium tin oxide (ITO) plate. The assembly process was characterized by advanced electrochemical and spectroscopic techniques. The bare ITO electrode generated three irreversible oxidation peaks, whereas the HSA-AuNC-modified electrode produced a pair of redox peaks for bilirubin at a formal potential of 0.27V (vs. Ag/AgCl). However, the native HSA protein immobilized on the ITO electrode failed to produce any redox peak for bilirubin. The results indicate that the AuNCs present in HSA act as electron transfer bridge between bilirubin and the ITO plate. Docking studies of AuNC with HSA revealed that the best docked structure of the nanocluster is located around the vicinity of the bilirubin binding site, with an orientation that allows specific oxidation. When the HSA-AuNC-modified electrode was employed for the detection of bilirubin using chronoamperometry at 0.3V (vs. Ag/AgCl), a steady-state current response against bilirubin in the range of 0.2μM to 7μM, with a sensitivity of 0.34μAμM(-1) and limit of detection of 86.32nM at S/N 3, was obtained. The bioelectrode was successfully applied to measure the bilirubin content in spiked serum samples. The results indicate the feasibility of using HSA-AuNC as a biorecognition element for the detection of serum bilirubin levels using an electrochemical technique.

  13. Study of interactions between oppositely charged dendrigraft poly-L-lysine and human serum albumin by continuous frontal analysis capillary electrophoresis and fluorescence spectroscopy.

    PubMed

    Sisavath, Nicolas; Leclercq, Laurent; Le Saux, Thomas; Oukacine, Farid; Cottet, Hervé

    2013-05-10

    Dendrigraft poly-L-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery systems, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aims at determining the number of binding sites and the corresponding successive equilibrium constants between DGL of generation 3 (G3) and HSA in physiological conditions. To meet this end, continuous frontal analysis capillary electrophoresis (FACCE) and fluorescence spectroscopic methods were implemented and compared. FACCE was performed on a polycationic modified capillary in combination with a co-pressure that allowed for selectively introducing the free G3 from the G3/HSA mixtures. FACCE studies demonstrated that HSA has 2 binding sites with DGL G3 with the following successive constants K1=31.2×10(3) M(-1) and K2=30.6×10(3) M(-1). For a 1 g/L concentration in G3 and assuming a plasmatic HSA concentration of 40g/L, these binding constants lead to only 5% free DGL in the medium. It was also shown that the interactions between G3 and HSA corresponded to a model of cooperative sites. These results are in good agreement with the presence of two negatively charged domains in the HSA. Good fitting of the fluorescence spectroscopy data was obtained using the equilibrium constants derived from FACCE. Nevertheless, due to the high number of fitting parameters, it was difficult to fit the fluorescence spectroscopic data independently of the results obtained by FACCE.

  14. Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)](-) (M = Ru, Os) complexes to human serum albumin.

    PubMed

    Dömötör, Orsolya; Rathgeb, Anna; Kuhn, Paul-Steffen; Popović-Bijelić, Ana; Bačić, Goran; Enyedy, Eva Anna; Arion, Vladimir B

    2016-06-01

    Overall binding affinity of sodium or indazolium cis/trans-[MCl4(1H-indazole)(NO)] (M = Ru, Os) complexes towards human serum albumin (HSA) and high molecular mass components of the blood serum was monitored by ultrafiltration. HSA was found to be mainly responsible for the binding of the studied ruthenium and osmium complexes. In other words, this protein can provide a depot for the compounds and can affect their biodistribution and transport processes. In order to elucidate the HSA binding sites tryptophan fluorescence quenching studies and displacement reactions with the established site markers warfarin and dansylglycine were performed. Conditional stability constants for the binding to sites I and II on HSA were computed showing that the studied ruthenium and osmium complexes are able to bind into both sites with moderately strong affinity (logK' = 4.4-5.1). Site I is slightly more favored over site II for all complexes. No significant differences in the HSA binding properties were found for these metal complexes demonstrating negligible influence of the type of counterion (sodium vs indazolium), the metal ion center identity (Ru vs. Os) or the position of the nitrosyl group on the binding event. Electron paramagnetic resonance spin labeling of HSA revealed that indazolium trans-[RuCl4(1H-indazole)(NO)] and long-chain fatty acids show competitive binding to HSA. Moreover, this complex has a higher affinity for site I, but when present in excess, it is able to bind to site II as well, and displace fatty acids. PMID:26908285

  15. Adsorption of an amphiphilic penicillin onto human serum albumin: characterisation of the complex.

    PubMed

    Ruso, J M; Taboada, P; Varela, L M; Attwood, D; Mosquera, V

    2001-08-30

    The complex formed by the interaction of the amphiphilic penicillin drug nafcillin and human serum albumin (HSA) in water at 25 degrees C has been characterised using a range of physicochemical techniques. Measurements of the solution conductivity and the electrophoretic mobility of the complexes have shown an ionic adsorption of the drug on the protein surface leading to a surface saturation at a nafcillin concentration of 0.012 mmol kg(-1) and subsequent formation of drug micelles in solutions of higher nafcillin concentration. Measurements of the size of the complex and the thickness of the adsorbed layer by static and dynamic light scattering have shown a gradual change in hydrodynamic radius of the complex with increasing drug concentration typical of a saturation rather than a denaturation process, the magnitude of the change being insufficient to account for any appreciable extension or unfolding of the HSA molecule. The interaction potential between the HSA/nafcillin complexes, and the stability of the complexes were determined from the dependence of diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug/protein complexes with an increase in the concentration of added drug.

  16. Evaluation of Ischemia-Modified Albumin, Malondialdehyde, and Advanced Oxidative Protein Products as Markers of Vascular Injury in Diabetic Nephropathy

    PubMed Central

    Ahmad, Afzal; Manjrekar, Poornima; Yadav, Charu; Agarwal, Ashish; Srikantiah, Rukmini Mysore; Hegde, Anupama

    2016-01-01

    AIM This study aimed at evaluation of ischemia-modified albumin (IMA), malondialdehyde (MDA), and advanced oxidative protein products (AOPP) as markers of vascular injury in diabetic nephropathy (DN) with derivation of cutoff values for the same. MATERIALS AND METHODS Study population comprised 60 diabetes patients and 30 controls, with diabetes patients further categorized into three groups based on urine albumin/creatinine ratio (UACR) of <30 mg/g (diabetes without microalbuminuria), 30–300 mg/g (early DN), and >300 mg/g of creatinine (overt DN). Serum IMA, MDA, and AOPP were estimated by enzyme-linked immunosorbent assay; HbA1c, serum creatinine, urine albumin, and urine creatinine were estimated using automated analyzers. Statistical analysis was done using analysis of variance, Pearson’s correlation coefficient, and receiver-operating characteristic curve. RESULTS A statistically significant difference was found in the levels of IMA among patients with early DN (154 ng/mL), diabetes without nephropathy (109.4 ng/mL), and healthy controls (45.7 ng/mL), with highest levels in early DN cases. Similar increase was seen in AOPP as well. A significant correlation was observed between IMA and UACR in diabetes without nephropathy (r = 0.448). CONCLUSION The present study postulates serum IMA as a novel biomarker for the assessment of disease progression in diabetes even before microalbuminuria, and a cutoff point ≥99 ng/mL can be used for detection of early DN. PMID:27158221

  17. Structure of the methyl orange-binding site on human serum albumin and its color-change mechanism.

    PubMed

    Ito, Shigenori; Yamamoto, Daisuke

    2015-01-01

    The goal in this study was to clarify the color-change mechanisms of methyl orange (MO) bound to human serum albumin (HSA) and the structure of the binding site. The absorbance of the MOHSA complex was measured at 560 nm in solutions of varying pH (pH 2.4-6.6). The obtained pH-dependent experimental data were consistent with the data calculated using the Henderson-Hasselbalch equation and pKa values (3.8, MO; 1.4, carboxyl group). The extent of the binding of MO to an HSA molecule was determined to be 1-4 by performing surface plasmon resonance analysis. Furthermore, the binding of MO to HSA was inhibited by warfarin. A fitting model of MO to HSA was created to evaluate these results based on PDB data (warfarin-HSA complex: 2BXD) and protein-structure analysis. The color-change mechanism of the MO-HSA complex appears to be as follows: the dissociated sulfo group of MO binds to Arg218/Lys444 sidechains through electrostatic interaction in the warfarin-binding site, and, subsequently, the color change occurs through a proton exchange between the diazenyl group and the γ-carboxyl group of Glu292. The color-changed MO is fixed in the warfarin-binding site. These results could support the development of a reliable dye-binding method and of a new method for staining diverse tissues that is based on a validated mechanism. PMID:26299483

  18. Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods

    NASA Astrophysics Data System (ADS)

    Dong, Chengyu; Ma, Shuying; Liu, Ying

    2013-02-01

    This study was designed to examine the interaction of demeclocycline (DMCTC) with human serum albumin (HSA) by multi-spectroscopic and molecular docking methods. The inner filter effect was corrected before we calculated the binding parameters. Fluorescence and UV-vis spectroscopy revealed that DMCTC induced the fluorescence quenching of HSA though a static quenching procedure. Thermodynamic analysis by Van Hoff equation found enthalpy change (ΔH) and entropy change (ΔS) were -53.01 kJ mol-1 and -65.13 J mol-1 K-1, respectively, which indicated hydrogen bond and van der Waals force were the predominant force in the binding process. According to fluorescence resonance energy transfer (FRET), the specific binding distances between Trp-214 (donor) and DMCTC (acceptor) were 3.18 nm. Through site marker competitive experiments, subdomain IIA of HSA has been assigned to possess the high-affinity binding site of DMCTC. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with DMCTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of α-helices content about 4.8%, β-sheet from 30.3% to 21.6% and with increases of β-turn from 15.6% to 22.2%. Furthermore, the binding details between DMCTC and HSA were further confirmed by molecular docking studies, which revealed that DMCTC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces and π-π interactions. Moreover, the coexist metal ions such as Al3+, Fe3+, Cu2+, Cr3+ and Cd2+ can decrease the binding constants of DMCTC-HSA.

  19. Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin

    PubMed Central

    Mizuma, Takashi; McDonagh, Antony F; Lin, Emil T; Benet, Leslie Z

    1999-01-01

    Aims To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. Methods F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. Results Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the wafarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. Conclusions The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage. PMID:10383564

  20. Structure of Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Ho, Joseph X.

    1994-01-01

    Because of its availability, low cost, stability, and unusual ligand-binding properties, serum albumin has been one of the mst extensively studied and applied proteins in biochemistry. However, as a protein, albumin is far from typical, and the widespread interest in and application of albumin have not been balanced by an understanding of its molecular structure. Indeed, for more than 30 years structural information was surmised based solely on techniques such as hydrodynamics, low-angle X-ray scattering, and predictive methods.

  1. [Study on the interaction of doxycycline with human serum albumin].

    PubMed

    Hu, Tao-Ying; Chen, Lin; Liu, Ying

    2014-05-01

    The present study was designed to investigate the interaction of doxycycline (DC) with human serum albumin (HSA) by the inner filter effects, displacement experiments and molecular docking methods, based on classic multi-spectroscopy. With fluorescence quenching method at 298 and 310 K, the binding constants Ka, were determined to be 2. 73 X 10(5) and 0. 74X 10(5) L mol-1, respectively, and there was one binding site between DC and HSA, indicating that the binding of DC to HSA was strong, and the quenching mechanism was a static quenching. The thermodynamic parameters (enthalpy change, AH and enthropy change, delta S) were calculated to be -83. 55 kJ mol-1 and -176. 31 J mol-1 K-1 via the Vant' Hoff equation, which indicated that the interaction of DC with HSA was driven mainly by hydrogen bonding and van der Waals forces. Based on the Föster's theory of non-radiation energy transfer, the specific binding distance between Trp-214 (acceptor) and DC (donor) was 4. 98 nm, which was similar to the result confirmed by molecular docking. Through displacement experiments, sub-domain IIA of HSA was assigned to possess the high-affinity binding site of DC. Three-dimensional fluorescence spectra indicated that the binding of DC to HSA induced the conformation change of HSA and increased the disclosure of some part of hydrophobic regions that had been buried before. The results of FTIR spectroscopy showed that DC bound to HSA led to the slight unfolding of the polypeptide chain of HSA. Furthermore, the binding details between DC and HSA were further confirmed by molecular docking methods, which revealed that DC was bound at sub-domain IIA through multiple interactions, such as hydrophobic effect, polar forces and pi-pi interactions. The experimental results provide theoretical basis and reliable data for the study of the interaction between small drug molecule and human serum albumin PMID:25095435

  2. Behaviors of bovine serum albumin and rapeseed proteins at the air/water interface after grafting aliphatic or aromatic chains.

    PubMed

    Gerbanowski, Alice; Rabiller, Claude; Guéguen, Jacques

    2003-06-15

    The influence of grafting aliphatic or aromatic groups on the behaviors of bovine serum albumin (BSA) and rapeseed proteins (napin and cruciferin) at the air/water interface is studied. From compression isotherms, it is shown that the chemical modification induces an increase in the interfacial molecular areas of the three proteins. The more hydrophobic the groups grafted, the more important this increase is. The dilatational modulus clearly emphasized that the grafting of hydrophobic groups also leads to an increase of the collapse pressure, demonstrating a higher cohesiveness and resistance to pressure of the interfacial films. These results are discussed on the basis of the physicochemical changes due to these chemical modifications, especially the conformation, the surface hydrophobicity, and the flexibility of the modified proteins. The improvement of surface properties obtained by grafting aliphatic or aromatic chains onto these proteins looks very promising in regard to emulsifying and foaming properties.

  3. Stabilization and immune response of HBsAg encapsulated within poly(lactic-co-glycolic acid) microspheres using HSA as a stabilizer.

    PubMed

    Xu, Wenjuan; He, Jintian; Wu, Guanghao; Xiong, Fangfang; Du, Huijuan; Wang, Gaizhen

    2015-12-30

    The aim of this study was to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing hepatitis B virus surface antigen (HBsAg) using human serum albumin (HSA) as a stabilizer. Lyophilization and emulsification of HBsAg solution with dichloromethane caused a considerable loss of HBsAg antigenicity. Thus, the effects of HSA and trehalose on HBsAg recovery during lyophilization and emulsification were investigated. Adding HSA to HBsAg solutions significantly improved antigen recovery to >90% during lyophilization and emulsification. The effects of co-encapsulated HSA on the characteristics of the PLGA microspheres and stability of HBsAg released from the microspheres were also investigated. The in vitro release test showed that HBsAg was released from the PLGA microspheres continuously over seventy days. A large amount of released HBsAg was inactive without co-encapsulation of HSA. On the contrary, with HSA co-encapsulation, the released HBsAg retained approximately 90% of its antigenicity. The single injection of the HBsAg-HSA-loaded PLGA microspheres in rats resulted in higher anti-HBsAg IgG and Th1 cytokine levels than the single injection of the HBsAg-loaded microspheres or two injections of the conventional aluminum-adjuvanted HBsAg vaccine. Based on these findings, the HBsAg-HSA-loaded PLGA microspheres could be an effective carrier for HBsAg and form a promising depot system.

  4. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs. PMID:27294530

  5. On-site preparation of technetium-99m labeled human serum albumin for clinical application.

    PubMed

    Wang, Yuh-Feng; Chuang, Mei-Hua; Chiu, Jainn-Shiun; Cham, Thau-Ming; Chung, Mei-Ing

    2007-04-01

    Technetium-99m labeled human serum albumin (Tc-99m HSA) is an important radiopharmaceutical for clinical applications, such as cardiac function tests or protein-losing gastroenteropathy assessment. However, because of transfusion-induced infectious diseases, the safety of serum products is a serious concern. In this context, serum products acquired from patients themselves are the most ideal tracer. However, the development of rapid separation and easy clinical labeling methods is not yet well established. Under such situation, products from the same ethnic group or country are now recommended by the World Health Organization as an alternative preparation. This article describes the on-site preparation of Tc-99m HSA from locally supplied serum products. Different formulations were prepared and the labeling efficiency and stability were examined. Radio-labeling efficiencies were more than 90% in all preparation protocols, except for one that omitted the stannous solution. The most cost-effective protocol contained HSA 0.1 mg, treated with stannous fluoride 0.2 mg, and mixed with Tc-99m pertechnetate 30 mCi. A biodistribution study was performed in rats using a gamma camera immediately after intravenous administration of radiolabeled HSA. Tissue/organ uptake was obtained by measuring the radioactivity in organs after sacrificing the rats at timed intervals. The biologic half-life was about 32 min, determined from sequential venous blood collections. These data indicate that our preparation of Tc-99m HSA is useful and potentially applicable clinically. In addition, this on-site preparation provides the possibility of labeling a patient's own serum for subsequent clinical application.

  6. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    PubMed

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

  7. Stimulation of albumin endocytosis by cationized ferritin in cultured aortic smooth muscle cells

    SciTech Connect

    Sprague, E.A.; Kelley, J.L.; Suenram, C.A.; Valente, A.J.; Abreu-Macomber, M.; Schwartz, C.J.

    1985-12-01

    Anionic microdomains within the aortic smooth muscle cell (SMC) surface glycocalyx represent a potential barrier to the endocytosis of anionic plasma proteins. Cultured SMCs exposed briefly to cationized ferritin (CF) exhibit ultrastructural aggregations of surface anionic sites resulting in intervening areas essentially devoid of anionic charge. Preincubation of cultured aortic medial SMCs with 0.2 mg/ml CF for 1 minute at 37 C resulted in a 4-fold increase in binding and a 13-fold increase in internalization of /sup 125/I-human serum albumin (/sup 125/I-HSA) relative to cells pretreated with native ferritin. When both the CF preincubation and the endocytosis were performed at 4 C, the influence of CF was abolished. Studies at 4 C indicated that CF pretreatment of SMC at 37 C induced high affinity (Kd = 1.5 nM) saturable /sup 125/I-HSA binding, in addition to low-affinity nonsaturable binding. These results were further confirmed by binding competition studies using increasing concentrations of unlabeled HSA. In contrast, low-density lipoprotein, a large anionic molecule, failed to compete with /sup 125/I-HSA for binding sites on CF-pretreated SMCs at either 4 or 37 C. Pulse-chase studies at 37 C indicated that 20-30% of internalized /sup 125/I-HSA was degraded, and 40-50% exocytosed within 24 hours in CF-treated cells. CF pretreatment of the SMCs did not significantly enhance the uptake of /sup 14/C-sucrose as a measure of fluid-phase endocytosis at 30 and 60 minutes. The results of these studies emphasize the potentially important regulatory roles of cell-surface anionic charge distribution and cationic molecules in cellular endocytosis.

  8. Biodegradable synthetic polymer scaffolds for reinforcement of albumin protein solders used for laser-assisted tissue repair.

    PubMed

    Hoffman, Grant T; Soller, Eric C; McNally-Heintzelman, Karen M

    2002-01-01

    Laser tissue soldering has been investigated for several years by researchers in our laboratory as an alternative to conventional tissue fasteners, including sutures, staples and clips. Laser tissue soldering is a bonding technique in which protein solder is applied to the tissue surfaces to be joined, and laser energy is used to bond the solder to the tissue surfaces. Over the past four years we have been investigating the use of synthetic polymer membranes as a means for reinforcing the strength of tissue repairs formed using traditional laser tissue soldering techniques. The purpose of this study was to assess the influence of various processing parameters on the strength of tissue repairs formed using the reinforced solder. Biodegradable polymer membranes of specific porosity were fabricated by means of a solvent-casting and particulate-leaching technique, using three different poly(alpha ester)s: polyglycolic acid (PGA), polylactic acid (PLA) and poly(L-lactic-co-glycolic acid) (PLGA). In addition, several membranes were also prepared with poly(ethylene glycol) (PEG). The membranes were then doped with the traditional protein solder mixture of serum albumin and indocyanine green dye. Varied processing parameters included the polymer type, the PLGA copolymer blend ratio, the polymer/PEG blend ratio, the porosity of the polymer membrane and the initial albumin weight fraction. Variation of the polymer type had negligible effect on the strength of the repairs. Although it is known that alteration of the copolymer blend ratio of PLGA influences the degradation rate of the polymer, this variation also had no significant effect on the strength of the repairs formed. Increased membrane flexibility was observed when PEG was added during the casting stage. An increase in the porosity of the polymer membranes led to a subsequent increase in the final concentration of protein contained within the membranes, hence aiding in strengthening the resultant repairs. Likewise

  9. The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model

    PubMed Central

    2013-01-01

    It is not exactly known why certain food proteins are more likely to sensitize. One of the characteristics of most food allergens is that they are stable to the acidic and proteolytic conditions in the digestive tract. This property is thought to be a risk factor in allergic sensitization. The purpose of the present study was to investigate the contribution of the protein structure of 2S albumin (Ber e1), a major allergen from Brazil nut, on the sensitizing capacity in vivo using an oral Brown Norway rat food allergy model. Disulphide bridges of 2S albumin were reduced and alkylated resulting in loss of protein structure and an increased pepsin digestibility in vitro. Both native 2S albumin and reduced/alkylated 2S albumin were administered by daily gavage dosing (0.1 and 1 mg) to Brown Norway rats for 42 days. Intraperitoneal administration was used as a positive control. Sera were analysed by ELISA and passive cutaneous anaphylaxis. Oral exposure to native or reduced/alkylated 2S albumin resulted in specific IgG1 and IgG2a responses whereas only native 2S albumin induced specific IgE in this model, which was confirmed by passive cutaneous anaphylaxis. This study has shown that the disruption of the protein structure of Brazil nut 2S albumin decreased the sensitizing potential in a Brown Norway rat food allergy model, whereas the immunogenicity of 2S albumin remained preserved. This observation may open possibilities for developing immunotherapy for Brazil nut allergy. PMID:24180644

  10. The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model.

    PubMed

    Van Bilsen, Jolanda Hm; Knippels, Léon Mj; Penninks, André H; Nieuwenhuizen, Willem F; De Jongh, Harmen Hj; Koppelman, Stef J

    2013-11-04

    : It is not exactly known why certain food proteins are more likely to sensitize. One of the characteristics of most food allergens is that they are stable to the acidic and proteolytic conditions in the digestive tract. This property is thought to be a risk factor in allergic sensitization. The purpose of the present study was to investigate the contribution of the protein structure of 2S albumin (Ber e1), a major allergen from Brazil nut, on the sensitizing capacity in vivo using an oral Brown Norway rat food allergy model. Disulphide bridges of 2S albumin were reduced and alkylated resulting in loss of protein structure and an increased pepsin digestibility in vitro. Both native 2S albumin and reduced/alkylated 2S albumin were administered by daily gavage dosing (0.1 and 1 mg) to Brown Norway rats for 42 days. Intraperitoneal administration was used as a positive control. Sera were analysed by ELISA and passive cutaneous anaphylaxis. Oral exposure to native or reduced/alkylated 2S albumin resulted in specific IgG1 and IgG2a responses whereas only native 2S albumin induced specific IgE in this model, which was confirmed by passive cutaneous anaphylaxis. This study has shown that the disruption of the protein structure of Brazil nut 2S albumin decreased the sensitizing potential in a Brown Norway rat food allergy model, whereas the immunogenicity of 2S albumin remained preserved. This observation may open possibilities for developing immunotherapy for Brazil nut allergy.

  11. Synthesis and Applications of Multimodal Hybrid Albumin Nanoparticles for Chemotherapeutic Drug Delivery and Photothermal Therapy Platforms

    NASA Astrophysics Data System (ADS)

    Peralta, Donna V.

    Progress has been made in using human serum albumin nanoparticles (HSAPs) as carrier systems for targeted treatment of cancer. Human serum albumin (HSA), the most abundant human blood protein, can form HSAPs via a desolvation and crosslinking method, with the size of the HSAPs having crucial importance for drug loading and in vivo performance. Gold nanoparticles have also gained medicinal attention due to their ability to absorb near-infrared (NIR) light. These relatively non-toxic particles offer combinational therapy via imaging and photothermal therapy (PPTT) capabilities. A desolvation and crosslinking approach was employed to encapsulate gold nanoparticles (AuNPs), hollow gold nanoshells (AuNSs), and gold nanorods (AuNRs), into efficiently sized HSAPs for future tumor heat ablation via PPTT. The AuNR-HSAPs, AuNP-HSAPs and AuNS-HSAPs had average particle diameters of 222 +/- 5, 195 +/- 9 and 156 +/- 15, respectively. We simultaneously encapsulated AuNRs and the anticancer drug paclitaxel (PAC), forming PAC-AuNR-HSAPs with overall average particle size of 299 +/- 6 nm. Loading of paclitaxel into PAC-AuNR-HSAPs reached 3microg PAC/mg HSA. PAC-AuNR-HSAPs experienced photothermal heating of 46 °C after 15 minutes of NIR laser exposure; the temperature necessary to cause severe cellular hyperthermia. There was a burst release of paclitaxel up to 188 ng caused by the irradiation session, followed by a temporal drug release. AuNR-HSAPs were tested for ablation of renal cell carcinoma using NIR irradiation in vitro. Particles created with the same amount of AuNRs, but varying HSA (1, 5 or 20 mg) showed overall particle size diameters 409 +/- 224, 294 +/- 83 and 167 +/- 4 nm, respectively. Increasing HSAPs causes more toxicity under non-irradiated treatment conditions: AuNR-HSAPs with 20 mg versus 5 mg HSA caused cell viability of 64.5% versus 87%, respectively. All AuNR-HSAPs batches experienced photothermal heating above 42 °C. Coumarin-6, was used to visualize the

  12. CHARACTERIZATION OF INTERACTION KINETICS BETWEEN CHIRAL SOLUTES AND HUMAN SERUM ALBUMIN BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Tong, Zenghan; Hage, David S.

    2011-01-01

    Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2–9.6 s−1 for the two enantiomers of m-HPPH and 3.2–4.1 s−1 for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions. PMID:21872871

  13. Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations.

    PubMed

    Tatsumi, Akitoshi; Okada, Masaya; Inagaki, Yoshihiro; Inoue, Sachiyo; Hamaguchi, Tsuneo; Iwakawa, Seigo

    2016-01-01

    Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA. PMID:27476944

  14. Study on the interaction of levocetirizine dihydrochloride with human serum albumin by molecular spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Xiangping; Du, Yingxiang; Sun, Wen; Kou, Junping; Yu, Boyang

    2009-12-01

    The interaction between cetirizine dihydrochloride and human serum albumin (HSA) has been examined by the spectroscopic techniques first. According to Stern-Volmer equation at different temperatures and the UV-vis spectra examination it was demonstrated that HSA fluorescence quenching initiated by levocetirizine was static. The values of binding constant ( KA) and the number of binding sites ( n) for levocetirizine and HSA were smaller than those for cetirizine and HSA, which meant that the transport of drug was regulated by the stereoselectivity of HSA to the enantiomer. The effect of the non-enzymatic glycosylation (NEG) on the interaction between levocetirizine and HSA signified that the administration of levocetirizine for diabetes should be different from the normal. The positive Δ S° and negative Δ H° indicated that ionic interaction played a major role between levocetirizine and HSA. Circular dichroism (CD) measurement showed that the secondary structure of HSA has changed in the presence of levocetirizine, and α-helical content decreased from 63.1% for free HSA to 54.9% for combined HSA, and accordingly the other secondary structure (β-strand, β-turns and others) contents increased to some extent. Finally, by the competitive binding experiments it was deduced that levocetirizine specifically bound to HSA in the region of site II, which meant the curative effect of levocetirizine should be reconsidered when it was administrated together with other site II drugs.

  15. Oxidative Deamination of Serum Albumins by (-)-Epigallocatechin-3-O-Gallate: A Potential Mechanism for the Formation of Innate Antigens by Antioxidants

    PubMed Central

    Hatasa, Yukinori; Chikazawa, Miho; Furuhashi, Mai; Nakashima, Fumie; Shibata, Takahiro; Kondo, Tatsuhiko; Akagawa, Mitsugu; Hamagami, Hiroki; Tanaka, Hiroshi; Tachibana, Hirofumi; Uchida, Koji

    2016-01-01

    (-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies. PMID:27046229

  16. Effect of HSA coated iron oxide labeling on human umbilical cord derived mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Sanganeria, Purva; Chandra, Sudeshna; Bahadur, Dhirendra; Khanna, Aparna

    2015-03-01

    Human umbilical cord derived mesenchymal stem cells (hUC-MSCs) are known for self-renewal and differentiation into cells of various lineages like bone, cartilage and fat. They have been used in biomedical applications to treat degenerative disorders. However, to exploit the therapeutic potential of stem cells, there is a requirement of sensitive non-invasive imaging techniques which will offer the ability to track transplanted cells, bio-distribution, proliferation and differentiation. In this study, we have analyzed the efficacy of human serum albumin coated iron oxide nanoparticles (HSA-IONPs) on the differentiation of hUC-MSCs. The colloidal stability of the HSA-IONPs was tested over a long period of time (≥20 months) and the optimized concentration of HSA-IONPs for labeling the stem cells was 60 μg ml-1. Detailed in vitro assays have been performed to ascertain the effect of the nanoparticles (NPs) on stem cells. Lactate dehydrogenase (LDH) assay showed minimum release of LDH depicting the least disruptions in cellular membrane. At the same time, mitochondrial impairment of the cells was also not observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry analysis revealed lesser generation of reactive oxygen species in HSA-IONPs labeled hUC-MSCs in comparison to bare and commercial IONPs. Transmission electron microscopy showed endocytic engulfment of the NPs by the hUC-MSCs. During the process, the gross morphologies of the actin cytoskeleton were found to be intact as shown by immunofluorescence microscopy. Also, the engulfment of the HSA-IONPs did not show any detrimental effect on the differentiation potential of the stem cells into adipocytes, osteocytes and chondrocytes, thereby confirming that the inherent properties of stem cells were maintained.

  17. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    PubMed

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. PMID:25193649

  18. Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications.

    PubMed

    Gokara, Mahesh; Malavath, Tirupathi; Kalangi, Suresh Kumar; Reddana, Pallu; Subramanyam, Rajagopal

    2014-01-01

    Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45 μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of KAsA = 3.86 ± 0.01 × 10(4) M(-1), which corresponds to the free energy of (ΔG) -6.3 kcal M(-1) at 25 °C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50 ± 2.4 to 50% ± 2.3 and an increase in the β-turns from 25 ± 0.65 to 29% ± 0.91 and random coils from 17.5% ± 0.95 to 21% ± 1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA-AsA complexes. PMID:23844909

  19. Comparative methyl linoleate and methyl linolenate oxidation in the presence of bovine serum albumin at several lipid/protein ratios.

    PubMed

    Zamora, Rosario; Hidalgo, Francisco J

    2003-07-30

    The oxidation of methyl linoleate (LMe) and methyl linolenate (LnMe) in the presence of bovine serum albumin (BSA) in the dark at 60 degrees C was studied to analyze the role of the type of fatty acid and the protein/lipid ratio on the relative progression of the processes involved when lipid oxidation occurs in the presence of proteins. The disappearance of the fatty acid, the formation of primary and secondary products of lipid peroxidation, the loss of amino acid residues, the production of oxidized lipid/amino acid reaction products, and the development of color and fluorescence were studied as a function of incubation time in protein/lipid samples at 10:1, 6:1, and 3:1 w/w ratios. The incubation of LMe and LnMe in the presence of BSA at 60 degrees C rapidly produced lipid peroxidation and protein damage. Although reaction rates were much faster for LnMe than for LMe, both fatty acids had similar behaviors, and LnMe seemed to be only slightly more reactive than LMe for BSA by producing a higher increase of protein pyrroles in the protein and the development of increased browning and fluorescence. The protein/lipid ratio also influenced the relative progress of the reactions implicated. Thus, a lower protein/lipid ratio increased sample oxidation and protein damage. This also produced an increased browning, in accordance with the mechanisms proposed for browning production by oxidized lipid/protein reactions. On the contrary, browning of extracted lipids increased at higher protein/lipid ratios. This opposite tendency allowed evaluation of the overall significance of the different browning processes implicated in the final colors observed, concluding that color changes observed in BSA/lipid samples were mostly a consequence of oxidized lipid/protein reactions. PMID:14705893

  20. Mapping the interactions between the Alzheimer's Aβ-peptide and human serum albumin beyond domain resolution.

    PubMed

    Algamal, Moustafa; Milojevic, Julijana; Jafari, Naeimeh; Zhang, William; Melacini, Giuseppe

    2013-10-01

    Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aβ. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association. PMID:24094411

  1. On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study.

    PubMed

    Eleta-Lopez, Aitziber; Etxebarria, Juan; Reichardt, Niels-Christian; Georgieva, Radostina; Bäumler, Hans; Toca-Herrera, José L

    2015-10-01

    The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

  2. On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study.

    PubMed

    Eleta-Lopez, Aitziber; Etxebarria, Juan; Reichardt, Niels-Christian; Georgieva, Radostina; Bäumler, Hans; Toca-Herrera, José L

    2015-10-01

    The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption. PMID:26218522

  3. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  4. O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    SciTech Connect

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-03-04

    Research highlights: {yields} Human serum heme-albumin displays globin-like properties. {yields} O{sub 2}-mediated oxidation of ferrous nitrosylated human serum heme-albumin. {yields} Allosteric modulation of human serum heme-albumin reactivity. {yields} Rifampicin is an allosteric effector of human serum heme-albumin. {yields} Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O{sub 2}-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10{sup -5} and 8.3 x 10{sup -4} s{sup -1}, and h = 1.3 x 10{sup -4} and 8.5 x 10{sup -4} s{sup -1}, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 {sup o}C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O{sub 2}-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O{sub 2} does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O{sub 2}-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  5. Stereo-Selectivity of Human Serum Albumin to Enantiomeric and Isoelectronic Pollutants Dissected by Spectroscopy, Calorimetry and Bioinformatics

    PubMed Central

    Ahmad, Ejaz; Rabbani, Gulam; Zaidi, Nida; Singh, Saurabh; Rehan, Mohd; Khan, Mohd Moin; Rahman, Shah Kamranur; Quadri, Zainuddin; Shadab, Mohd.; Ashraf, Mohd Tashfeen; Subbarao, Naidu; Bhat, Rajiv; Khan, Rizwan Hasan

    2011-01-01

    1–naphthol (1N), 2–naphthol (2N) and 8–quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (Kb) of these pollutants to HSA were moderate (104–105 M−1). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39–5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy–entropy compensation (EEC). The difference observed between ΔCpexp and ΔCpcalc are suggested to be caused by binding–induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants. PMID:22073150

  6. Protein-based nanotubes for biomedical applications

    NASA Astrophysics Data System (ADS)

    Komatsu, Teruyuki

    2012-03-01

    This review presents highlights of our latest results of studies directed at developing protein-based smart nanotubes for biomedical applications. These practical biocylinders were prepared using an alternate layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into a nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-l-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)3]. The obtained (PLA/HSA)3 nanotubes (NTs) can be dispersed in aqueous medium and are hydrated significantly. Several ligands for HSA, such as zinc(ii) protoporphyrin IX (ZnPP), were bound to the HSA component in the cylindrical wall. Similar NTs comprising recombinant HSA mutant, which has a strong binding affinity for ZnPP, captured the ligand more tightly. The Fe3O4-coated NTs can be collected easily by exposure to a magnetic field. The hybrid NTs bearing a single avidin layer as an internal wall captured biotin-labeled nanoparticles into the central channel when their particle size is sufficiently small to enter the pores. The NTs with an antibody surface interior entrapped human hepatitis B virus with size selectivity. It is noteworthy that the infectious Dane particles were encapsulated completely into the hollows. Other HSA-based NTs having an α-glucosidase inner wall hydrolysed a glucopyranoside to yield α-d-glucose. A perspective of the practical use of the protein-based NTs is also described.

  7. Studies on the binding of a carditionic agent to human serum albumin by two-dimensional correlation fluorescence spectroscopy and molecular modeling

    NASA Astrophysics Data System (ADS)

    Wang, Teng; Xiang, Bing-Ren; Li, Yue; Chen, Chang-Yun; Zhou, Xiao-Hua; Wang, Zhi-Mei; Dong, Ying; Wang, Ying; Fang, Hui-Sheng

    2009-03-01

    The binding of a novel carditionic agent (2-amino-4-chloro- N-(4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)benzamide (ACPB)) to human serum albumin (HSA) under physiological conditions has been investigated by using UV/vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) in combination with protein-ligand docking study. It was observed that there was a prominent interaction between ACPB and HSA. The interaction was also confirmed by two-dimensional (2D) correction analysis based on the quenching perturbation on the fluorescence spectra and the order of the response of ACPB and HSA to the quenching perturbation was also elucidated based on Noda's rule. Fluorescence data revealed that the fluorescence quenching was a static quenching process and the binding constants were calculated to be 8.781 × 10 5, 7.310 × 10 5, and 5.358 × 10 5 M -1 at 290, 300, and 310 K, respectively. The thermodynamic parameters were calculated according to the Van't Hoff equation and the binding mode was determined. In addition, the alterations of protein secondary structure were qualitatively and quantitatively determined by the evidence from synchronous fluorescence, CD and FT-IR. Furthermore, docking studies that corroborate our experimental results revealed that the binding sites were located in subdomain IIA of HSA.

  8. Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin.

    PubMed

    Nunthanawanich, Pornpimon; Sompong, Weerachat; Sirikwanpong, Sukrit; Mäkynen, Kittana; Adisakwattana, Sirichai; Dahlan, Winai; Ngamukote, Sathaporn

    2016-01-01

    Advanced glycation end products (AGEs) play an important factor for pathophysiology of diabetes and its complications. Moringa oleifera is one of the medicinal plants that have anti-hyperglycemic activity. However, anti-glycation property of Moringa oleifera leaf extract on the different types of reducing monosaccharides-induced protein glycation has not been investigated. Therefore, the aim of this study was to examine the protective effect of Moringa oleifera aqueous leaf extract (MOE) on reducing sugars-induced protein glycation and protein oxidation. Total phenolic content of MOE was measured using the Folin-Ciocalteu method. Bovine serum albumin was incubated with 0.5 M of reducing sugars (glucose or fructose) with or without MOE (0.5-2.0 mg/mL) for 1, 2, 3 and 4 weeks. The results found that total phenolic content was 38.56 ± 1.50 mg gallic acid equivalents/g dry extract. The formation of fluorescent and non-fluorescent AGEs [N (ε)-(carboxymethyl) lysine (CML)] and the level of fructosamine were determined to indicate protein glycation, whereas the level of protein carbonyl content and thiol group were examined for protein oxidation. MOE (0.5-2.0 mg/mL) significantly inhibited the formation of fluorescent, N (ε)-CML and markedly decreased fructosamine level (P < 0.05). Moreover, MOE significantly prevented protein oxidation manifested by reducing protein carbonyl and the depletion of protein thiol in a dose-dependent manner (P < 0.05). Thus, the findings indicated that polyphenols containing in MOE have high potential for decreasing protein glycation and protein oxidation that may delay or prevent AGE-related diabetic complications. PMID:27468399

  9. Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin.

    PubMed

    Nunthanawanich, Pornpimon; Sompong, Weerachat; Sirikwanpong, Sukrit; Mäkynen, Kittana; Adisakwattana, Sirichai; Dahlan, Winai; Ngamukote, Sathaporn

    2016-01-01

    Advanced glycation end products (AGEs) play an important factor for pathophysiology of diabetes and its complications. Moringa oleifera is one of the medicinal plants that have anti-hyperglycemic activity. However, anti-glycation property of Moringa oleifera leaf extract on the different types of reducing monosaccharides-induced protein glycation has not been investigated. Therefore, the aim of this study was to examine the protective effect of Moringa oleifera aqueous leaf extract (MOE) on reducing sugars-induced protein glycation and protein oxidation. Total phenolic content of MOE was measured using the Folin-Ciocalteu method. Bovine serum albumin was incubated with 0.5 M of reducing sugars (glucose or fructose) with or without MOE (0.5-2.0 mg/mL) for 1, 2, 3 and 4 weeks. The results found that total phenolic content was 38.56 ± 1.50 mg gallic acid equivalents/g dry extract. The formation of fluorescent and non-fluorescent AGEs [N (ε)-(carboxymethyl) lysine (CML)] and the level of fructosamine were determined to indicate protein glycation, whereas the level of protein carbonyl content and thiol group were examined for protein oxidation. MOE (0.5-2.0 mg/mL) significantly inhibited the formation of fluorescent, N (ε)-CML and markedly decreased fructosamine level (P < 0.05). Moreover, MOE significantly prevented protein oxidation manifested by reducing protein carbonyl and the depletion of protein thiol in a dose-dependent manner (P < 0.05). Thus, the findings indicated that polyphenols containing in MOE have high potential for decreasing protein glycation and protein oxidation that may delay or prevent AGE-related diabetic complications.

  10. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  11. [Influence of direct electric current on hydrodynamic diameter of human serum albumin].

    PubMed

    Korpan, M I; Gorchev, V F; Chekman, I S; Gun'ko, V M; Fialko-Moser, V

    2008-01-01

    The effect of the weak electric current (2 mA/cm2) on structural characteristics (hydrodynamic diameter and molecular weight) of the human serum albumin (HSA) was studied using photon correlation spectroscopy (PCS). The average diameter of initial HSA globule is approximately 7 nm (66.8 kDa). After electric current treatment during 2-5 min the diameter of HSA monomer increases to 7.5 nm. The duration of electric current treatment being increased to 20 min the size of HSA monomers decreases to 6.4 nm. The behaviour of HSA oligomers is close to that of monomers. Consequently, changes in the sizes of monomers and oligomers of HSA under the electric current treatment are caused by the change in the charge density stimulating change of tertiary structure of molecules and possible addition of ions from the buffer solution to them.

  12. Interaction of diuron to human serum albumin: Insights from spectroscopic and molecular docking studies.

    PubMed

    Chen, Huilun; Rao, Honghao; Yang, Jian; Qiao, Yongxiang; Wang, Fei; Yao, Jun

    2016-01-01

    This investigation was undertaken to determine the interaction of diuron with human serum albumin (HSA) was studied by monitoring the spectral behavior of diuron-HSA system. The fluorescence of HSA at 340 nm excited at 230 nm was obviously quenched by diuron due to dynamic collision and the quenching constant was of the order of 10(4) L mol(-1) at 310 K. However, no fluorescence quenching was observed when excited at 280 nm. Thermodynamic investigations revealed that the combination between diuron and HSA was entropy driven by predominantly hydrophobic interactions. The binding of diuron induced the drastic reduction in α-helix conformation and the significant enhancement in β-turn conformation of HSA. In addition, both sites marker competition study and molecular modeling simulation evidenced the binding of diuron to HSA primarily took place in subdomain IIIA (Sudlow's site II). PMID:26671830

  13. In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique.

    PubMed

    Davilas, A; Koupparis, M; Macheras, P; Valsami, G

    2006-11-01

    The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients. PMID:17132209

  14. Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

    PubMed

    Pasternak, Kamila; Nowacka, Olga; Wróbel, Dominika; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

    2014-03-01

    The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

  15. Doxorubicin-loaded nanoparticles consisted of cationic- and mannose-modified-albumins for dual-targeting in brain tumors.

    PubMed

    Byeon, Hyeong Jun; Thao, Le Quang; Lee, Seunghyun; Min, Sun Young; Lee, Eun Seong; Shin, Beom Soo; Choi, Han-Gon; Youn, Yu Seok

    2016-03-10

    Albumin nanoparticles have been increasingly viewed as an effective way of delivering chemotherapeutics to solid tumors. Here, we report the one-pot development of a unique prototype of doxorubicin-loaded nanoparticles (NPs) made of naïve albumin (HSA) plus cationic- (c-HSA) or mannose-modified-albumin (m-HSA), with the goal of traversing the blood-brain barrier and targeting brain tumors. c-HSA was synthesized by conjugating ethylenediamine to naïve HSA. Then, m-HSA was derivatized using mannopyranoside via a thiol-maleimide reaction. The c/m-HSA NPs were prepared using a mixture solution of c- and m-HSAs in deionized water and doxorubicin in ethanol/chloroform in the same pot using a high-pressure homogenizer. The c/m-HSA NPs were spherical and well-dispersed, with a particle size of 90.5±3.1nm and zeta-potential of -12.0±0.3mV at c- and m-HSA feed ratios of 5% and 10%, respectively. The c/m-HSA NPs displayed good stability over 3days based on particle size and a linear gradual doxorubicin release over 2days. Specifically, the inhibitory concentration (IC50; 0.5±0.02μg/ml) of c/m-HSA NPs was >2.2-15.6 fold lower than those of doxorubicin or the other HSA NPs. Moreover, among HSA NPs, c/m-HSA NPs exhibited the most prominent performances in transport across the bEnd.3 cell monolayer and uptake in bEnd.3 cells as well as U87MG glioblastoma cells and spheroids. Furthermore, c/m-HSA NPs were localized to a greater extent in brain glioma compared to naïve HSA NPs. Orthotopic glioma-bearing mice treated with c/m-HSA NPs displayed significantly smaller tumors than the mice treated with saline, doxorubicin or HSA NPs. This improved anti-glioma efficacy seemed to be due to the dual-enhanced system of dual cationic absorptive transcytosis and glucose-transport by the combined use of c- and m-HSAs. The c/m-HSA NPs have potential as a novel anti-brain cancer agent with good targetability. PMID:26826308

  16. Interaction of ionic detergent cethyltrimethylammonium bromide with human serum albumin at various values of pH: Spectroscopic study

    NASA Astrophysics Data System (ADS)

    Vlasova, Irina M.; Vlasov, Alexander A.; Saletsky, Alexander M.

    2010-12-01

    The interaction of cationic detergent cethyltrimethylammonium bromide (CTAB) with human serum albumin (HSA) at various values of pH has been studied using steady-state tryptophan fluorescence of HSA, polarized tryptophan fluorescence of HSA, fluorescence of nanomarker eosin in solutions of HSA and CTAB and Raman spectroscopy. By methods of fluorescent analysis the qualitative rearrangements of HSA globules at denaturation under action of CTAB are registered, whereas by Raman spectroscopy the quantitative changes of secondary structure of HSA at CTAB-induced denaturation are determined. It is shown that denaturation of HSA, taking place at interaction of cationic detergent CTAB with HSA, has one-stage character. At interaction of CTAB with HSA the deepest denaturation of HSA is reached at concentration of 4 mM CTAB (in the range of pH 3.5-8.0). More intensive denaturation of HSA under action of CTAB takes place at values of pH, higher than the isoelectric point of HSA (pI 4.7).

  17. Bioactivity of albumins bound to silver nanoparticles.

    PubMed

    Mariam, Jessy; Sivakami, S; Kothari, D C; Dongre, P M

    2014-06-01

    The last decade has witnessed a tremendous rise in the proposed applications of nanomaterials in the field of medicine due to their very attractive physiochemical properties and novel actions such as the ability to reach previously inaccessible targets such as brain. However biological activity of functional molecules bound to nanoparticles and its physiological consequences is still unclear and hence this area requires immediate attention. The functional properties of Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) bound to silver nanoparticles (~60 nm) have been studied under physiological environment. Esterase activity, binding of drugs (warfarin and ibuprofen), antioxidant activity and copper binding by albumins was evaluated. The catalytic efficiencies of HSA and BSA diminished upon binding to silver nanoparticles. Perturbation in binding of warfarin and ibuprofen, loss of free sulphydryls, antioxidant activity and enhancement of copper binding were observed in albumins bound to nanoparticles. These alterations in functional activity of nanoparticle bound albumins which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.

  18. Binding interaction of a gamma-aminobutyric acid derivative with serum albumin: an insight by fluorescence and molecular modeling analysis.

    PubMed

    Pal, Uttam; Pramanik, Sumit Kumar; Bhattacharya, Baisali; Banerji, Biswadip; C Maiti, Nakul

    2016-01-01

    gamma-Aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter and some of its derivatives showed potential to act as neuroprotective agents. With the aim of developing potential leads for anti-Alzheimer's drugs, in this study we synthesized a novel GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate by a unique method of Buchwald-Hartwig cross coupling synthesis; with some modification the yield was significant (97 %) and spectroscopic analysis confirmed that the compound was highly pure (98.8 % by HPLC). The druglikeness properties such as logP, logS, and polar surface area were 3.87, -4.86 and 94.17 Å(2) respectively and it satisfied the Lipinski's rule of five. We examined the binding behavior of the molecule to human serum albumin (HSA) and bovine serum albumin (BSA) which are known as universal drug carrier proteins. The molecule binds to the proteins with low micromolar efficiency and the calculated binding constants were 3.85 and 2.75 micromolar for BSA and HSA, respectively. Temperature dependent study using van't Hoff equation established that the binding was thermodynamically favorable and the changes in the Gibb's free energy, ΔG for the binding process was negative. However, the binding of the molecule to HSA was enthalpy driven and the change of enthalpy (ΔH) was -10.63 kJ/mol, whereas, the binding to BSA was entropy driven and the change in entropy ΔS was 222 J/mol. The molecular docking analysis showed that the binding sites of the molecule lie in the groove between domain I and domain III of BSA, whereas it is within the domain I in case of HSA, which also supported the different thermodynamic nature of binding with HSA and BSA. Molecular dynamics analysis suggested that the binding was stable with time and provided further details of the binding interaction. Molecular dynamics study also highlighted the effect of this ligand binding on the serum albumin structure. PMID

  19. Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

    SciTech Connect

    Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

    2008-01-01

    The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors.

  20. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  1. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands

    PubMed Central

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-01-01

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock. PMID:26853907

  2. Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants.

    PubMed

    Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

    2015-02-25

    Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200×10(-5) mol L(-1). In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

  3. Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants

    NASA Astrophysics Data System (ADS)

    Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

    2015-02-01

    Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200 × 10-5 mol L-1. In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

  4. Poly-S-Nitrosated Albumin as a Safe and Effective Multifunctional Antitumor Agent: Characterization, Biochemistry and Possible Future Therapeutic Applications

    PubMed Central

    Ishima, Yu; Kragh-Hansen, Ulrich; Maruyama, Toru; Otagiri, Masaki

    2013-01-01

    Nitric oxide (NO) is a ubiquitous molecule involved in multiple cellular functions. Inappropriate production of NO may lead to disease states. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated human serum albumin (Mono-SNO-HSA), and that is why we are testing whether this albumin form can be therapeutically useful. Recently, we developed SNO-HSA analogs such as SNO-HSA with many conjugated SNO groups (Poly-SNO-HSA) which were prepared using chemical modification. Unexpectedly, we found striking inverse effects between Poly-SNO-HSA and Mono-SNO-HSA. Despite the fact that Mono-SNO-HSA inhibits apoptosis, Poly-SNO-HSA possesses very strong proapoptotic effects against tumor cells. Furthermore, Poly-SNO-HSA can reduce or perhaps completely eliminate the multidrug resistance often developed by cancer cells. In this review, we forward the possibility that Poly-SNO-HSA can be used as a safe and effective multifunctional antitumor agent. PMID:24490156

  5. Rate constant for reaction of vitamin C with protein radicals in γ-irradiated aqueous albumin solution at 295 K

    NASA Astrophysics Data System (ADS)

    Miyazaki, Tetsuo; Yoshimura, Toru; Mita, Kazuya; Suzuki, Keiji; Watanabe, Masami

    1995-02-01

    When an aqueous solution of albumin (0.1 kg dm -3) is irradiated by γ-rays at 295 K, albumin radicals with a long lifetime are observed by ESR. The reaction of vitamin C with the albumin radicals has been studied at 295 K in the albumin solution, which is considered as a model of cells. The rate constant for the reaction of vitamin C with the albumin radicals was measured as 0.014 dm 3 mol -1 s -1, which is much smaller than the reported rate constants (10 6-10 10 dm 3 mol -1 s -1) for the reaction of vitamin C with radicals in a dilute aqueous solution. The small rate constant for the reaction of vitamin C is ascribed to the reaction in polymer coils in the albumin solution, since vitamin C and albumin radicals diffuse very slowly in the coils.

  6. Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.

    PubMed

    Basuli, Falguni; Li, Changhui; Xu, Biying; Williams, Mark; Wong, Karen; Coble, Vincent L; Vasalatiy, Olga; Seidel, Jurgen; Green, Michael V; Griffiths, Gary L; Choyke, Peter L; Jagoda, Elaine M

    2015-03-01

    We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent.

  7. Synthesis of Fluorine-18 Radio-labeled Serum Albumins for PET Blood Pool Imaging

    PubMed Central

    Basuli, Falguni; Li, Changhui; Xu, Biying; Williams, Mark; Wong, Karen; Coble, Vincent L; Vasalatiy, Olga; Seidel, Jurgen; Green, Michael V.; Griffiths, Gary L.; Choyke, Peter L.; Jagoda, Elaine M.

    2015-01-01

    We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [18F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [18F]tetrabutylammonium fluoride ([18F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [18F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH 9) for 15 min at 37–40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18–35% (n = 30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [18F]RSA is an effective blood pool imaging agent in rats and might, as [18F]HSA, prove similarly useful as a clinical imaging agent. PMID:25533724

  8. Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery

    PubMed Central

    Mehtala, Jonathan G.; Kulczar, Chris; Lavan, Monika; Knipp, Gregory; Wei, Alexander

    2015-01-01

    Polyethylene glycol (PEG) derivatives were conjugated onto the Cys-34 residue of human serum albumin (HSA) to determine their effects on the solubilization, permeation, and cytotoxic activity of hydrophobic drugs such as paclitaxel (PTX). PEG(C34)HSA conjugates were prepared on a multigram scale by treating native HSA (n-HSA) with 5- or 20-kDa mPEG-maleimide, resulting in up to 77% conversion of the mono-PEGylated adduct. Nanoparticle tracking analysis of PEG(C34)HSA formulations in phosphate buffer revealed an increase in nanosized aggregates relative to n-HSA, both in the absence and presence of PTX. Cell viability studies conducted with MCF-7 breast cancer cells indicated that PTX cytotoxicity was enhanced by PEG(C34)HSA when mixed at 10:1 mole ratios, up to a two-fold increase in potency relative to n-HSA. The PEG(C34)HSA conjugates were also evaluated as PTX carriers across monolayers of HUVEC and hCMEC/D3 cells, and found to have nearly identical permeation profiles as n-HSA. PMID:25918947

  9. Recent Topics in Chemical and Clinical Research on Glycated Albumin

    PubMed Central

    Ueda, Yuki; Matsumoto, Hideyuki

    2015-01-01

    The measuring method for glycated albumin (GA) has been developed as a new glycemic control marker since the beginning of the 21st century. Since GA has an advantage in reflecting glycemic status over a shorter period than hemoglobin A1c (HbA1c), much research and many reviews have been reported. However, so far there have been few reports on glycation sites based on the tertiary structure of human serum albumin (HSA) and the comparison of glycation rates between GA and HbA1c in detail. The present review discusses how the glycation sites of lysine residues in HSA are modified with glucose, whereas the glycation sites of lysine residues are located inside of HSA as well as the direct comparison of glycation rates between GA and HbA1c using human blood. Moreover, the most recent clinical researches on GA are described. PMID:25614014

  10. Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

    PubMed

    Chudzik, Mariola; Równicka-Zubik, Joanna; Pożycka, Jadwiga; Pawelczak, Bartosz; Sulkowska, Anna

    2016-01-01

    Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.

  11. The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

    NASA Astrophysics Data System (ADS)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

    2012-04-01

    Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

  12. Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin*

    PubMed Central

    Ascenzi, Paolo; di Masi, Alessandra; Coletta, Massimo; Ciaccio, Chiara; Fanali, Gabriella; Nicoletti, Francesco P.; Smulevich, Giulietta; Fasano, Mauro

    2009-01-01

    Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role

  13. Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein.

    PubMed

    Yeggoni, Daniel Pushpa Raju; Manidhar, Darla Mark; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2016-09-01

    Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be KUmb-1=3.1 ± .01 × 10(4) M(-1), KUmb-2 = 7 ± .01 × 10(4) M(-1), which corresponds to -6.1 and -6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of KUmb-1-AGP=3.1 ± .01 × 10(3) M(-1) and KUmb-2-AGP = 4.6 ± .01 × 10(3) M(-1). Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster's theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1-HSA and Umb-2-HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.

  14. Albumin Test

    MedlinePlus

    ... to a variety of conditions in addition to malnutrition , a decrease in albumin needs to be evaluated ... can also be seen in inflammation , shock, and malnutrition . They may be seen with conditions in which ...

  15. Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma.

    PubMed

    Johannesson, Gunvor A; Kristiansson, Monica H; Jönsson, Bo A G; Lindh, Christian H

    2008-03-01

    An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.

  16. Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

    PubMed

    Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

    2013-01-01

    We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

  17. Caffeine and sulfadiazine interact differently with human serum albumin: A combined fluorescence and molecular docking study

    NASA Astrophysics Data System (ADS)

    Islam, Mullah Muhaiminul; Sonu, Vikash K.; Gashnga, Pynsakhiat Miki; Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2016-01-01

    The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0 × 104 M-1) in comparison with CAF (∼9.3 × 102 M-1) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure.

  18. Albuminated Glycoenzymes: Enzyme Stabilization through Orthogonal Attachment of a Single-Layered Protein Shell around a Central Glycoenzyme Core.

    PubMed

    Ritter, Dustin W; Newton, Jared M; Roberts, Jason R; McShane, Michael J

    2016-05-18

    Here we demonstrate an approach to stabilize enzymes through the orthogonal covalent attachment of albumin on the single-enzyme level. Albuminated glycoenzymes (AGs) based upon glucose oxidase and catalase from Aspergillus niger were prepared in this manner. Gel filtration chromatography and dynamic light scattering support modification, with an increase in hydrodynamic radius of ca. 60% upon albumination. Both AGs demonstrate a marked resistance to aggregation during heating to 90 °C, but this effect is more profound in albuminated catalase. The functional characteristics of albuminated glucose oxidase vary considerably with exposure type. The AG's thermal inactivation is reduced more than 25 times compared to native glucose oxidase, and moderate stabilization is observed with one month storage at 37 °C. However, albumination has no effect on operational stability of glucose oxidase.

  19. 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.

  20. Comparison of photoluminescence properties of HSA-protected and BSA-protected Au25 nanoclusters

    NASA Astrophysics Data System (ADS)

    Tsukamoto, Masato; Kawasaki, Hideya; Saitoh, Tadashi; Inada, Mitsuru; Kansai Univ. Collaboration

    Gold nanoclusters (NCs) have attracted great interest for a wide range of applications. In particular, red light-emitting Au25 NCs have been prepared with various biological ligands. It has been shown that Au25 NCs have Au13-core/6Au2(SR)3-semiring structure. The red luminescence thought to be originated from both core (670 nm) and semiring (625 nm). It is important to reveal a structure of Au25 NCs to facilitate the progress of applications. However, the precise structure of Au25 NCs has not been clarified. There is a possibility of obtaining structural information about Au25 NCs to compare optical properties of the NCs that protected by slightly different molecules. Bovine and human serum albumin (BSA, HSA) are suitable one for this purpose. It has been suggested that rich tyrosine and cysteine residues in these molecules are important to produce the thiolate-protected Au NCs. If Au25 NCs have core/shell structure, only the luminescence of the semiring will be affected by the difference of the albumin molecules. We carefully compared PL characteristics of BSA- and HSA- protected Au25 NCs. As a result, there was no difference in the PL at 670 nm (core), while differences were observed in the PL at 625 nm (semiring). The results support that Au25 NCs have core/semiring structure.

  1. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    PubMed Central

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  2. Ultraviolet-circular dichroism spectroscopy and potentiometric study of the interaction between human serum albumin and sodium perfluorooctanoate.

    PubMed

    Messina, Paula; Prieto, Gerardo; Dodero, Verónica; Ruso, Juan M; Schulz, Pablo; Sarmiento, Félix

    2005-12-15

    The interaction of a fluorinated surfactant, sodium perfluorooctanoate, with human serum albumin (HSA) has been investigated by a combination of ultraviolet-circular dichroism (UV-CD) spectroscopy and potentiometry (by a home-built ion-selective electrode) techniques to detect and characterize the conformational transitions of HSA. By using difference spectroscopy, the transition was followed as a function of temperature, and the data were analyzed to obtain the parameters characterizing the thermodynamics of unfolding. The results indicate that the presence of surfactant drastically changes the melting unfolding, acting as a structure stabilizer and delaying the unfolding process. Potentiometric measurements were used to determine the binding isotherms and binding capacity for this system. The isotherm shows a high affinity of surfactant molecules for HSA. The average number of surfactant molecules absorbed per protein molecule (at 28 mM of surfactant concentration) was found to be approximately 900, about 6 g of surfactant per gram of protein. The shape of the binding capacity curve and the relation between binding capacity and extend of cooperativity were examined. From these analysis, the values of g (number of ligand-binding sites), KH (Hill binding constant), and nH (Hill coefficient) were determined.

  3. Targeting of naproxen covalently linked to HSA to sinusoidal cell types of the liver.

    PubMed

    Melgert, B N; Wartna, E; Lebbe, C; Albrecht, C; Molema, G; Poelstra, K; Reichen, J; Meijer, D K

    1998-01-01

    The kinetic behaviour of a naproxen human serum albumin conjugate (Nap23-HSA) was investigated in rats and in isolated perfused rat livers (IPRL), as compared to its active metabolite naproxen-lysine (Nap-lysine) and free naproxen. Through covalently linking the anti-inflammatory drug naproxen to HSA, this drug can be selectively delivered to non parenchymal cells of the liver. Liver endothelial and Kupffer cells play an important role in the pathogenesis of inflammatory liver diseases. Targeting naproxen to these cells might increase its efficacy and reduce the side effects. The altered kinetic properties of Nap23-HSA, after i.v. injection of 22 mg x kg(-1), as compared to an equimolar amount of the uncoupled drug, were demonstrated in vivo by a decrease in the steady state volume of distribution (41 +/- 5 vs. 134 +/- 19 ml x kg(-1)), a decrease in its clearance (0.48 +/- 0.05 vs. 0.63 +/- 0.1 ml x min(-1) x kg(-1)), a shorter plasma half life (60 +/- 11 vs. 152 +/- 44 min) and a sustained biliary excretion. Liver targeting of Nap23-HSA was clearly demonstrated: drug content of the liver 180 min after injection was about 30 times higher for Nap23-HSA as compared to naproxen itself. The IPRL experiments showed that the Vmax of hepatic removal of the conjugate was 40 microg x min(-1) x g liver(-1). With doses below receptor saturation a rapid removal of the conjugate (t1/2 = 6 min) from the perfusion medium was found. In conclusion, this study demonstrates the saturable uptake of Nap23-HSA and its lysosomal degradation in both in vivo and IPRL experiments. Covalently linked naproxen is released as Nap-lysine. This active metabolite accumulates in Kupffer and endothelial cells in which it reaches therapeutic concentrations. Release from these cells leads to rapid uptake by hepatocytes and carrier mediated excretion into bile. Levels of Nap-lysine in bile and plasma reflect the slowest step in its generation: the proteolytic release in endothelial and Kupffer cells.

  4. Role of hsa-miR-325 in the etiopathology of preeclampsia.

    PubMed

    Lázár, Levente; Nagy, Bálint; Molvarec, Attila; Szarka, András; Rigó, János

    2012-09-01

    Preeclampsia (PE) is a common pregnancy-specific syndrome characterized by hypertension and proteinuria. Evidence has demonstrated that hypertensive disorders in pregnancy are associated with alterations in the expression of different microRNAs (miRNAs). miRNAs are endogenously expressed non-coding RNAs that have significant biological and pathological functions due to their potential mechanisms of regulation of gene expression. The purpose of the present study was to investigate the expression of hsa-miR-325 in placental samples of preeclamptic and uncomplicated pregnancy patients. hsa-miR-325 was isolated from placenta tissue samples obtained from 31 preeclamptic and 28 normotensive pregnant females. Quantitative real-time polymerase chain reaction was used to analyze miRNA expression. The expression of hsa-miR‑325 was elevated in uncomplicated pregnancies compared with preeclamptic patients. ΔCt (mean±SD) values were 0.117±0.07 in PE tissues and 0.135±0.051 in normotensive cases (p<0.05). The expression levels correlated with patient blood pressure (p=0.015, r=-0.23), and tended to correlate with body mass index (p=0.065, r=0.261). The expression of hsa-miR-325 was downregulated in the case of PE. Changes in hsa-miR‑325 expression in the case of pregnancy-related hypertensive disorders might affect the oxidative stress pathways and heat-shock protein production. These factors have a strong correlation with the development of PE. We, therefore, suggest that hsa-miR-325 contributes to the pathogenesis of PE. PMID:22710575

  5. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures

    PubMed Central

    Miranda, Érica G. A.; Tofanello, Aryane; Brito, Adrianne M. M.; Lopes, David M.; Albuquerque, Lindomar J. C.; de Castro, Carlos E.; Costa, Fanny N.; Giacomelli, Fernando C.; Ferreira, Fabio F.; Araújo-Chaves, Juliana C.; Nantes, Iseli L.

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3–12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with

  6. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures.

    PubMed

    Miranda, Érica G A; Tofanello, Aryane; Brito, Adrianne M M; Lopes, David M; Albuquerque, Lindomar J C; de Castro, Carlos E; Costa, Fanny N; Giacomelli, Fernando C; Ferreira, Fabio F; Araújo-Chaves, Juliana C; Nantes, Iseli L

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the

  7. Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures.

    PubMed

    Miranda, Érica G A; Tofanello, Aryane; Brito, Adrianne M M; Lopes, David M; Albuquerque, Lindomar J C; de Castro, Carlos E; Costa, Fanny N; Giacomelli, Fernando C; Ferreira, Fabio F; Araújo-Chaves, Juliana C; Nantes, Iseli L

    2016-01-01

    The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the

  8. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  9. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    PubMed

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.

  10. Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

    PubMed Central

    Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

    2013-01-01

    Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

  11. Protein adsorption to multi-component glasses

    NASA Astrophysics Data System (ADS)

    Hall, Matthew Micah

    2003-07-01

    The adsorption of human serum albumin (HSA) to sodium silicate, soda lime silicate (SLS), and sodium aluminosilicate (SAS) glass microspheres was investigated using sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with a colloidal silver stain for visualization. The 30 Na2O·70 SiO2 composition could not be evaluated due to an apparent chemical interference that occurred during silver staining. This inhibitory effect was attributed to the extensive corrosion that occurred during the protein elution and caused an elevation in the pH of the solution. The remaining glass compositions were sufficiently durable for further study. The HSA adsorption capacity of SLS glass microspheres containing 70 and 80 mol% SiO2 increased as CaO was substituted for Na2O. An abrupt decrease in the HSA adsorption capacity was observed for SLS glasses containing 60 mol% SiO2. A similar trend was observed for the SAS glass microspheres, although the SAS glasses adsorbed less HSA than the SLS glasses containing equivalent molar percentages of SiO2. The initial increase in HSA adsorption capacity for SLS and SAS glasses containing 70 and 80 MOM SiO2 was attributed to the introduction of positive charges into the glass surfaces via Ca2+ and Al3+ cations. The decrease in HSA adsorption capacity for SLS and SAS glasses containing 60 mol% SiO2 may be due to an enhanced affinity between the glasses and HSA, resulting in a "flattened" conformation that limits the total accessible area for adsorption.

  12. [Albumin in sepsis].

    PubMed

    Tamion, F

    2010-09-01

    Human serum albumin is a small (66kD) globular protein representing over 60 % of the total plasma protein content. It is made up of 585 amino 6 acids and contains 35 cysteine residues forming disulfide bridges that contribute to its overall tertiary structure. It has a free cysteine-derived thiol group at Cys-34, which accounts for 80 % of its redox activity. Physiologically, serum albumin exists in a reduced form with a free thiol contributing to its antioxidant properties. It is synthesized primarily in the liver and is an acute-phase protein. It is a multifunctional plasma protein ascribed ligand-binding and transport properties as well as antioxidants and enzymatic functions. It maintains colloid osmotic pressure, modulates inflammatory response and may influence oxidative damage. Hypoalbuminemia is common in the intensive care unit and may be due to decreased synthesis by the liver and/or to increased losses or increased proteolysis and clearance. Although albumin was long used to control vascular collapse in critically ill patients, the evidence suggests that it does not offer a benefit over crystalloid solutions in vascular collapse. However, human serum albumin is an important circulating antioxidant and it may be beneficial in critically ill patients to limit oxidative damage. A number of studies suggest that in specific groups of hypoalbuminemic critically ill patients, albumin administration may have beneficial effects on organ function, although the exact mechanisms remain undefined. Further trials are needed to confirm theses observations and to clearly demonstrate whether albumin should be administered in critically ill patients with hypoalbuminemia. PMID:20675098

  13. A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

    PubMed

    Andrási, Melinda; Lehoczki, Gábor; Nagy, Zoltán; Gyémánt, Gyöngyi; Pungor, András; Gáspár, Attila

    2015-06-01

    This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M).

  14. A novel prototype of albumin nanoparticles fabricated by supramolecular cyclodextrin-adamantane association.

    PubMed

    Lee, Seunghyun; Lee, Changkyu; Kim, Bomi; Thao, Le Quang; Lee, Eun Seong; Kim, Jong Oh; Oh, Kyung Taek; Choi, Han-Gon; Youn, Yu Seok

    2016-11-01

    Albumin has been viewed as one of the most attractive biomacromolecules for making nanoparticulate systems due to its biocompatibility and chemical functionality. Thus far, albumin nanoparticles (NPs) are prepared by several limited methods, such as, desolvation, emulsification or high-pressure homogenization. In this article, we introduce a new albumin NPs prototype fabricated via a 'host' (β-cyclodextrin)-'guest' (adamantane) supramolecular association. These NPs (GC-CD/HSA-ADA NPs) consisted of β-cyclodextrin-modified glycol chitosan (GC-CD) and adamantane-conjugated human serum albumin (HSA-ADA) (GC-CD/HSA-ADA NPs) that were facilely prepared by a consequent dropwise mixing and sonication method. Doxorubicin-loaded GC-CD/HSA-ADA NPs exhibited an appropriate particle size (∼260nm), good physicochemical stability (∼48h), significant HCT116 cell cytotoxicity (IC50: 0.32μg/ml) and cell internalization. Furthermore, GC-CD/HSA-ADA NPs showed excellent tumor targetability probably due to gp60-mediated transcytosis mechanism because it was markedly accumulated in the tumor site of a HCT116 cell-xenograft mouse. Based on these results, these albumin NPs will be promising for a new NP platform that can be applied for cancer therapy or imaging. PMID:27522557

  15. Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.

    PubMed

    Alarcón-Segovia, D; Fishbein, E; Estrada-Parra, S; García-Ortigoza, E

    1976-03-01

    Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same

  16. Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein.

    PubMed

    Bista, Sudeep Raj; Haywood, Alison; Hardy, Janet; Lobb, Michael; Tapuni, Angela; Norris, Ross

    2015-03-01

    1.Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80-85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2.PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3.Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86-89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4.UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

  17. Bovine serum albumin (BSA) can replace patient serum as a protein source in an in vitro fertilization (IVF) program.

    PubMed

    Benadiva, C A; Kuczynski-Brown, B; Maguire, T G; Mastoianni, L; Flickinger, G L

    1989-06-01

    Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures. During an 11-month period 127 patients treated for in vitro fertilization had either their serum (N = 71) or bovine serum albumin (BSA; N = 56) used as the protein source in the insemination and growth media. Ham's F-10 + 0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media. Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively. Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups. Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA. Likewise, rates of abnormal fertilization, cleavage, and pregnancy were similar in both groups. In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%). Cleavage rates for the two groups were similar and the percentage of embryos developed to greater than or equal to 4 cells did not differ significantly. We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.

  18. Malondialdehyde, carbonyl proteins and albumin-disulphide as useful oxidative markers in mild cognitive impairment and Alzheimer's disease.

    PubMed

    Greilberger, J; Koidl, C; Greilberger, M; Lamprecht, M; Schroecksnadel, K; Leblhuber, F; Fuchs, D; Oettl, K

    2008-07-01

    The question arises as to whether oxidative stress has a primary role in neurodegeneration or is a secondary end-stage epiphenomenon. The aim of the present study was to determine oxidative stress parameters like malondialdehyde (MDA), carbonyl proteins (CP) and Albumin-disulphide (Alb-SSR) and relate these parameters to the immune parameter neopterin, folic acid and vitamin B12 as vitamins and homocysteine in patients with neuro-degenerative diseases (NDD), namely mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to an aged matched control group. MDA, CP and Alb-SSR were significantly increased in the NDD group compared to controls, but not vitamin B12, folic acid and neopterin. Significant correlations were found between CP and Alb-SSR, CP and MDA and between MDA and Alb-SSR including patients with NDD and the control group. These results support the hypothesis that oxidative damage to lipids and proteins is an important early event in the pathogenesis of neurodegenerative diseases.

  19. Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations.

    PubMed

    Zhou, Chen; Qi, Wei; Lewis, E Neil; Carpenter, John F

    2015-03-01

    A Raman spectrometer and dynamic light scattering system were combined in a single platform (Raman-DLS) to provide concomitant higher order structural and hydrodynamic size data for therapeutic proteins at high concentration. As model therapeutic proteins, we studied human serum albumin (HSA) and intravenous immunoglobulin (IVIG). HSA concentration and temperature interval during heating did not affect the onset temperatures for conformation perturbation or aggregation. The impact of pH on thermal stability of HSA was tested at pHs 3, 5, and 8. Stability was the greatest at pH 8, but distinct unfolding and aggregation behaviors were observed at the different pHs. HSA structural transitions and aggregation kinetics were also studied in real time during isothermal incubations at pH 7. In a forced oxidation study, it was found that hydrogen peroxide (H2O2) treatment reduced the thermal stability of HSA. Finally, the structure and thermal stability of IVIG were studied, and a comprehensive characterization of heating-induced structural perturbations and aggregation was obtained. In conclusion, by providing comprehensive data on protein tertiary and secondary structures and hydrodynamic size during real-time heating or isothermal incubation experiments, the Raman-DLS system offers unique physical insights into the properties of high-concentration protein samples. PMID:25475399

  20. [The interaction of vitamin B6 with the human serum albumin].

    PubMed

    Wang, Jian-lin; Fu, Lian-chun; Zhou, Shi-wu; Chen, Zhi-jian; Lu, Wen-bo; Ye, Xue-min; Meng, Guang-zheng; Song, Zeng-fu

    2005-06-01

    The Human Serum Albumin (HSA) can emit fluorescence (lamda(em) = 350 nm) under irradiation of ultraviolet light (lamda(ex) = 296 nm). After the vitamin B6 (B6) was added into HSA solution the fluorescence of HSA was quenched partially. This quenching was static quenching owing to the fact that ro (fluorescence life time (life time of the molecule excitation state) of HSA without B6) equaled to tau(i) (fluorescence life time of HSA with B6). The formation constant K between HSA and B6 was observed from the experiment, and then the binding distance R0 was determined (R0 = 1.872 nm). The CD spectra of samples (HSA, [HSA] = 1 x 10(-5) mol x L(-1), HSA +B6, [B6] = 5 x 10(-8), 1.5 x 10(-7), 2.5 x 10(-7), 3.5x 10(-7), 4.5x 10(-7) mol x L(-1)) were measured, and all CD spectra were almost the same. From [theta] values, the contents (percent content) of four structure(alpha-Helix, beta-Pleated, Beta-corner, and Randon wind) of samples can be calculated, and we find that all samples contain almost the same structure contents. PMID:16201370

  1. Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin.

    PubMed

    Arya, Shruti; Kumari, Arpana; Dalal, Vijit; Bhattacharya, Mily; Mukhopadhyay, Samrat

    2015-09-21

    The self-assembly of proteins triggered by a conformational switch into highly ordered β-sheet rich amyloid fibrils has captivated burgeoning interest in recent years due to the involvement of amyloids in a variety of human diseases and a diverse range of biological functions. Here, we have investigated the mechanism of fibrillogenesis of human serum albumin (HSA), an all-α-helical protein, using an array of biophysical tools that include steady-state as well as time-resolved fluorescence, circular dichroism and Raman spectroscopy in conjunction with atomic force microscopy (AFM). Investigations into the temporal evolution of nanoscale morphology using AFM revealed the presence of ring-like intermediates that subsequently transformed into worm-like fibrils presumably by a ring-opening mechanism. Additionally, a multitude of morphologically-diverse oligomers were observed on the pathway to amyloid formation. Kinetic analysis using multiple structural probes in-tandem indicated that HSA amyloid assembly is a concerted process encompassing a major structural change that is primarily mediated by hydrophobic interactions between thermally-induced disordered segments originating in various domains. A slower growth kinetics of aggregates suggested that the protein structural reorganization is a prerequisite for fibril formation. Moreover, time-dependent Raman spectroscopic studies of HSA aggregation provided key molecular insights into the conformational transitions occurring within the protein amide backbone and at the residue-specific level. Our data revealed the emergence of conformationally-diverse disulfides as a consequence of structural reorganization and sequestration of tyrosines into the hydrophobic amyloid core comprising antiparallel cross β-sheets.

  2. Study on the interaction of artificial and natural food colorants with human serum albumin: A computational point of view.

    PubMed

    Masone, Diego; Chanforan, Céline

    2015-06-01

    Due to the high amount of artificial food colorants present in infants' diets, their adverse effects have been of major concern among the literature. Artificial food colorants have been suggested to affect children's behavior, being hyperactivity the most common disorder. In this study we compare binding affinities of a group of artificial colorants (sunset yellow, quinoline yellow, carmoisine, allura red and tartrazine) and their natural industrial equivalents (carminic acid, curcumin, peonidin-3-glucoside, cyanidin-3-glucoside) to human serum albumin (HSA) by a docking approach and further refinement through atomistic molecular dynamics simulations. Due to the protein-ligand conformational interface complexity, we used collective variable driven molecular dynamics to refine docking predictions and to score them according to a hydrogen-bond criterion. With this protocol, we were able to rank ligand affinities to HSA and to compare between the studied natural and artificial food additives. Our results show that the five artificial colorants studied bind better to HSA than their equivalent natural options, in terms of their H-bonding network, supporting the hypothesis of their potential risk to human health. PMID:25935119

  3. Study of the effect of Cal-Red on the secondary structure of human serum albumin by spectroscopic techniques

    NASA Astrophysics Data System (ADS)

    Dong, Lijun; Chen, Xingguo; Hu, Zhide

    2007-11-01

    The effect of Cal-Red on the structure of human serum albumin (HSA) was studied using Resonance light scattering (RLS), Fourier transformed Infrared (FT-IR) and Circular dichroism (CD) spectroscopic methods. The RLS spectroscopic results show that the RLS intensity of HSA was significantly increased in the presence of Cal-Red. The binding parameters of HSA with Cal-Red were studied at different temperatures of 289, 299, 309 and 319 K at pH 4.1. It is indicated by the Scatchard plots that the binding constant K decreased from 4.03 × 10 8 to 7.59 × 10 7 l/mol and the maximum binding number N decreased from 215 to 152 with increasing the temperature, respectively. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The enthalpy change Δ H0, the free energy change Δ G0 and the entropy change Δ S0 of 289 K were calculated to be -42.75 kJ/mol, -47.56 kJ/mol and 16.66 J/mol K, respectively. The alterations of protein secondary structure in the presence of Cal-Red in aqueous solution were quantitatively calculated from FT-IR and CD spectroscopy with reductions of α-helices content about 5%, β-turn from 10% to 2% and with increases of β-sheet from 38% to 51%.

  4. Studies on the synthesis, characterization, human serum albumin binding and biological activity of single chain surfactant-cobalt(III) complexes.

    PubMed

    Vignesh, G; Sugumar, K; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The interaction of surfactant-cobalt(III) complexes [Co(bpy)(dien)TA](ClO4)3 · 3H2O (1) and [Co(dien)(phen)TA](ClO4)3 · 4H2O (2), where bpy = 2,2'-bipyridine, dien = diethylenetriamine, phen = 1,10-phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (Kb ) and number of binding-sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (∆G°, ∆H° and ∆S°) and Ea were also obtained. According to Förster's non-radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. PMID:26250655

  5. Electron microscopy imaging of proteins on gallium phosphide semiconductor nanowires

    NASA Astrophysics Data System (ADS)

    Hjort, Martin; Bauer, Mikael; Gunnarsson, Stefan; Mårsell, Erik; Zakharov, Alexei A.; Karlsson, Gunnel; Sanfins, Elodie; Prinz, Christelle N.; Wallenberg, Reine; Cedervall, Tommy; Mikkelsen, Anders

    2016-02-01

    We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the laminin conformation on the NWs. In blood plasma, an intermediate sized corona around the NWs indicates a corona with a mixture of plasma proteins. The ability to directly visualize proteins on nanostructures in situ holds great promise for assessing the conformation and thickness of the protein corona, which is key to understanding and predicting the properties of engineered nanomaterials in a biological environment.We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the

  6. Norfloxacin and N-Donor Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin Interaction, and Anti-Trypanosoma cruzi Activity

    PubMed Central

    Martins, Darliane A.; Gouvea, Ligiane R.; Muniz, Gabriel S. Vignoli; Louro, Sonia R. W.; Batista, Denise da Gama Jaen; Soeiro, Maria de Nazaré C.; Teixeira, Letícia R.

    2016-01-01

    Copper(II) complexes with the first-generation quinolone antibacterial agent norfloxacin containing a nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy) or 1,10-phenanthroline (phen) were prepared and characterized by IR, EPR spectra, molar conductivity, and elemental analyses. The experimental data suggest that norfloxacin was coordinated to copper(II) through the carboxylato and ketone oxygen atoms. The interaction of the copper(II) complexes with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated using fluorescence quenching of the tryptophan residues and copper(II) EPR spectroscopy. The results of fluorescence titration revealed that copper(II) complexes have a moderate ability to quench the intrinsic fluorescence of the albumins through a static quenching mechanism. EPR experiments showed that BSA and HSA Cu(II) sites compete with NOR for Cu(II)-bipy and Cu(II)-phen to form protein mixed-ligand complexes. Copper(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The tests performed using bloodstream trypomastigotes showed that the Cu(II)-N-donor precursors and the metal complexes were more active than the free fluoroquinolone. PMID:26924953

  7. Time-insensitive fluorescent sensor for human serum albumin and its unusual red shift.

    PubMed

    Smith, Sara E; Williams, Jessica M; Ando, Shin; Koide, Kazunori

    2014-03-01

    The concentration of human serum albumin (HSA) indicates the health state of individuals and is routinely measured by UV spectroscopy with bromocresol. However, this method tends to overestimate HSA, and more critically, depends highly on the timing, in seconds, of the measurements. Here, we report an analog of 2',7'-dichlorofluorescein that can be used as a fluorescent sensor to quantify HSA in human sera. The accuracy of this new method proved superior to that of bromocresol when an international standard serum sample was analyzed. This method is more convenient than the bromocresol method because it allows for fluorescence measurements during a >15 min period. Colorimetric analysis was also performed to further investigate the effects of the binding of the sensor to HSA. These spectroscopic studies suggest that absorption and emission changes upon HSA binding may be due to the dehydration of the dye and/or stabilization of the tritylic cation species.

  8. Binding of caffeine, theophylline, and theobromine with human serum albumin: A spectroscopic study

    NASA Astrophysics Data System (ADS)

    Zhang, Hong-Mei; Chen, Ting-Ting; Zhou, Qiu-Hua; Wang, Yan-Qing

    2009-12-01

    The interaction between three purine alkaloids (caffeine, theophylline, and theobromine) and human serum albumin (HSA) was investigated using UV/vis absorption, circular dichroism (CD), fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that three alkaloids caused the fluorescence quenching of HSA by the formation of alkaloid-HSA complex. The binding site number n and apparent binding constant KA, corresponding thermodynamic parameters the free energy change (Δ G), enthalpy change (Δ H), and entropy change (Δ S) at different temperatures were calculated. The hydrophobic interaction plays a major role in stabilizing the complex. The distance r between donor (HSA) and acceptor (alkaloids) was obtained according to fluorescence resonance energy transfer. The effect of alkaloids on the conformation of HSA was analyzed using circular dichroism (CD), UV/vis absorption, synchronous fluorescence and three-dimensional fluorescence spectra techniques.

  9. Binding properties of drospirenone with human serum albumin and lysozyme in vitro

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Ma, Xiangling; He, Jiawei; Sun, Qiaomei; Li, Yuanzhi; Li, Hui

    2016-01-01

    The interaction of drospirenone (DP) with human serum albumin (HSA)/lysozyme (LYZ) was investigated using different optical techniques and molecular models. Results from the emission and time resolved fluorescence studies revealed that HSA/LYZ emission quenching with DP was initiated by static quenching mechanism. The LYZ-DP system was more easily influenced by temperature than the HSA-DP system. Displacement experiments demonstrated that the DP binding site was mainly located in site 1 of HSA. Based on the docking methods, DP was mainly bound in the active site hinge region where Trp-62 and Trp-63 are located. Conformation study showed that DP had different effects on the local conformation of HSA and LYZ molecules.

  10. Low protein catabolic rate and serum albumin correlate with increased mortality and abdominal complications in peritoneal dialysis patients.

    PubMed

    Germain, M; Harlow, P; Mulhern, J; Lipkowitz, G; Braden, G

    1992-01-01

    We retrospectively reviewed 167 consecutive peritoneal dialysis patients with regard to serum albumin (Alb), mortality and abdominal complications. In addition, 25 patients were studied with serial measurements of urea kinetics. The patients were divided into four groups based on their dialysis index (DI) and normalized protein catabolic rate (NPCR) (Table I). 12/167 patients were identified with abdominal catastrophes. Before these complications occurred, the M Alb in this group was 2.67 + 0.24 (compared to age, sex and disease matched controls of 3.55 + .11 P < .05). Six of these patients died from abdominal complications. In the 26 patients with serial urea kinetic studies, 4/11 patients in group IV died (low NPCR and low DI) (P < .05 compared to Group I, II or III). We conclude that urea kinetic modeling is predictive of outcome in those patients with presumed poor nutrition and inadequate dialysis and that abdominal catastrophes are more common in those patients with poor nutrition. Prospective interventional studies should be designed in an attempt to improve the poor outcome in this group of patients.

  11. Synthesis of nano-bioactive glass-ceramic powders and its in vitro bioactivity study in bovine serum albumin protein

    NASA Astrophysics Data System (ADS)

    Nabian, Nima; Jahanshahi, Mohsen; Rabiee, Sayed Mahmood

    2011-07-01

    Bioactive glasses and ceramics have proved to be able to chemically bond to living bone due to the formation of an apatite-like layer on its surface. The aim of this work was preparation and characterization of bioactive glass-ceramic by sol-gel method. Nano-bioglass-ceramic material was crushed into powder and its bioactivity was examined in vitro with respect to the ability of hydroxyapatite layer to form on the surface as a result of contact with bovine serum albumin (BSA) protein. The obtained nano-bioactive glass-ceramic was analyzed before and after contact with BSA solution. This study used scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis to examine its morphology, crystallinity and composition. The TEM images showed that the NBG particles size were 10-40 nm. Bioactivity of nanopowder was confirmed by SEM and XRD due to the presence of a rich bone-like apatite layer. Therefore, this nano-BSA-bioglass-ceramic composite material is promising for medical applications such as bone substitutes and drug carriers.

  12. Preparation and characterization of bovine serum albumin surface-imprinted thermosensitive magnetic polymer microsphere and its application for protein recognition.

    PubMed

    Li, Xiangjie; Zhang, Baoliang; Li, Wei; Lei, Xingfeng; Fan, Xinlong; Tian, Lei; Zhang, Hepeng; Zhang, Qiuyu

    2014-01-15

    A novel bovine serum albumin surface-imprinted thermosensitive magnetic composite microsphere was successfully prepared by the surface grafting copolymerization method in the presence of temperature-sensitive monomer N-isopropylacrylamide (NIPAM), functional monomer methacrylic acid (MAA) and cross-linking agent N,N'-methylenebisacrylamide (MBA). The structure and component of the thermosensitive magnetic molecularly imprinted microsphere were investigated by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA). The results of thermosensitivity, adsorption capacity, selectivity and reusability showed the formation of a thermosensitivity grafting polymer layer P(NIPAM-MAA-MBA) on the surface of Fe3O4@SiO2 and the good adsorption capacity and specific recognition for template protein. When the adsorption temperature was higher than the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM), shape memory effect of imprinted cavities would be more effective. In other words, it was more conducive to capture template molecules under this condition and the imprinting factor would be higher. On the other hand, when the desorption temperature was lower than LCST of PNIPAM, the decrease of shape memory effect between imprinted cavities and template molecules would facilitate the release of template molecules from the imprinted cavities. Based on this property, the adsorption and desorption of template molecules could be regulated by system temperature indirectly which benefited from the existence of thermosensitivity imprinting layer.

  13. Resonance energy transfer, pH-induced folded states and the molecular interaction of human serum albumin and icariin.

    PubMed

    Cheng, Xiao-Xia; Fan, Xiao-Yang; Jiang, Feng-Lei; Liu, Yi; Lei, Ke-Lin

    2015-11-01

    Icariin is a flavonol glycoside with a wide range of pharmacological and biological activities. The pharmacological and biological functions of flavonoid compounds mainly originate from their binding to proteins. The mode of interaction of icariin with human serum albumin (HSA) has been characterized by fluorescence spectroscopy and far- and near-UV circular dichroism (CD) spectroscopy under different pH conditions. Fluorescence quenching studies showed that the binding affinity of icariin with HSA in the buffer solution at different pH values is: Ka (pH 4.5) > Ka (pH 3.5) > Ka (pH 9.0) > Ka (pH 7.0). Red-edge excitation shift (REES) studies revealed that pH had an obvious effect on the mobility of the tryptophan microenvironment and the addition of icariin made the REES effect more distinct. The static quenching mechanism and number of binding sites (n ≈ 1) were obtained from fluorescence data at three temperatures (298, 304 and 310 K). Both ∆H(0) < 0 and ∆Ѕ(0) < 0 suggested that hydrogen bonding and van der Waal's interaction were major driving forces in the binding mechanism, and this was also confirmed by the molecular simulation results. The distance r between the donor (HSA) and the acceptor (icariin) was calculated based on Förster non-radiation energy transfer theory. We found that pH had little impact on the energy transfer between HSA and icariin. Far- and near-UV CD spectroscopy studies further indicated the influence of pH on the complexation process and the alteration in the protein conformation upon binding.

  14. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

    PubMed

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-15

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

  15. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

    NASA Astrophysics Data System (ADS)

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-01

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

  16. Effect of hydration on interstitial distribution of charged albumin in rat dermis in vitro

    PubMed Central

    Wiig, Helge; Tenstad, Olav; Bert, Joel L

    2005-01-01

    At physiological pH, negatively charged glycosaminoglycans in the extracellular matrix may influence distribution volume of macromolecular probes, a phenomenon of importance for hydration of the interstitium and therefore for body fluid balance. We hypothesized that such charge effect was dependent on hydration. Human serum albumin (HSA) (the pH value for the isoelectric point (pI) = 4.9) was made neutral by cationization (cHSA) (pI = 7.6). Rat dermis was studied in vitro in a specially designed equilibration cell allowing control of hydration. Using a buffer containing labelled native HSA and cHSA, the distribution volumes were calculated relative to that of 51Cr-EDTA, an extracellular tracer. During changes in hydration (H), defined as (wet weight – dry weight) (dry weight)−1), the slope of the equation describing the relationship between extracellular fluid volume (Vx) (in g H2O (g dry weight)−1) and H (Vx = 0.925 H + 0.105) differed significantly from that for available volumes of cHSA (Va,cHSA = 0.624 H – 0.538) and HSA (Va,HSA = 0.518 H – 0.518). A gradual reduction in H led to a reduction in difference between available volumes for the two albumin species. Screening the fixed charges by 1 m NaCl resulted in similar available and excluded volumes of native HSA and neutral cHSA. We conclude that during gradual dehydration, there is a reduced effect of fixed negative charges on interstitial exclusion of charged macromolecules. This effect may be explained by a reduced hydration domain surrounding tissue and probe macromolecules in conditions of increased electrostatic interactions. Furthermore, screening of negative charges suggested that hyaluronan associated with collagen may influence intrafibrillar volume of collagen and thereby available and excluded volume fraction. PMID:16210353

  17. Real-Time Trapping of Intact Singly-Charged Bovine Serum Albumin Proteins with a Big Frequency-Adjusted Quadrupole

    SciTech Connect

    Koizumi, Hideya; Whitten, William B; Reilly, Pete

    2008-01-01

    High-resolution real-time particle mass measurements have not been achievable because the enormous amount of kinetic energy imparted to the particles upon expansion into vacuum competes with and overwhelms the forces applied to the charged particles within the mass spectrometer. It is possible to reduce the kinetic energy of a collimated particulate ion beam through collisions with a buffer gas while radially constraining their motion using a quadrupole guide or trap over a limited mass range. Controlling the pressure drop of the final expansion into a quadrupole trap permits a much broader mass range at the cost of sacrificing collimation. To achieve high-resolution mass analysis of massive particulate ions, an efficient trap with a large tolerance for radial divergence of the injected ions was developed that permits trapping a large range of ions for on-demand injection into an awaiting mass analyzer. The design specifications required that frequency of the trapping potential be adjustable to cover a large mass range and the trap radius be increased to increase the tolerance to divergent ion injection. The large-radius linear quadrupole ion trap was demonstrated by trapping singly-charged bovine serum albumin ions for on-demand injection into a mass analyzer. Additionally, this work demonstrates the ability to measure an electrophoretic mobility cross section (or ion mobility) of singly-charged intact proteins in the low-pressure regime. This work represents a large step toward the goal of high-resolution analysis of intact proteins, RNA, DNA, and viruses.

  18. Interaction of tetramethylpyrazine with two serum albumins by a hybrid spectroscopic method

    NASA Astrophysics Data System (ADS)

    Cheng, Zhengjun

    The interactions of tetramethylpyrazine (TMPZ) with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by various spectroscopic techniques. Fluorescence tests showed that TMPZ could bind to BSA/HSA to form complexes. The binding constants of TMPZ-BSA and TMPZ-HSA complexes were observed to be 1.442 × 104 and 3.302 × 104 M-1 at 298 K, respectively. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of TMPZ-HSA was mainly depended on hydrophobic interaction, and yet the binding of TMPZ-BSA might involve hydrophobic interaction strongly and electrostatic interaction. The results of synchronous fluorescence, three-dimensional fluorescence, UV-vis absorption, FT-IR and CD spectra showed that the conformations of both BSA and HSA altered with the addition of TMPZ. The binding average distance between TMPZ and BSA/HSA was evaluated according to Föster non-radioactive energy transfer theory. In addition, with the aid of site markers (such as, phenylbutazone, ibuprofen and digitoxin), TMPZ primarily bound to tryptophan residues of BSA/HSA within site I (sub-domain II A).

  19. Reviewing the binding of a series of parabens to human serum albumin.

    PubMed

    Greige-Gerges, Hélène; Kaissi, Rana; Magdalou, Jacques; Jraij, Alia

    2013-04-01

    To better understand the factors that contribute to the accumulation of unmetabolized parabens (p-hydroxybenzoic acid esters) in breast cancer tissue, the binding of a series of parabens (methyl-, ethyl-, butyl-, benzyl-paraben) to human serum albumin (HSA) was investigated by fluorescence spectroscopy and also their ability to modify the binding parameters of albumin site markers. Emission spectra of HSA upon fluorescence excitation of Trp 214 residue at 295 nm were recorded at different molar ratios of PB/HSA and data were corrected for the inner-filter effect. A significant inner-filter effect was obtained for molar ratios of 2.0 and above. For lower molar ratios, a slight increase in fluorescence of HSA was detected. p-Hydroxybenzoic acid, the main metabolite of parabens, did not modify the fluorescence of HSA whatever the molar ratio used. Binding parameters for compounds that are markers of site I, bilirubin and warfarin, were determined in the absence and presence of methyl, butyl and benzyl paraben at molar ratios of PB/HSA of 0, 1 and 2. No variation of the binding constants of these markers was observed. The results indicate that parabens weakly interact with HSA thus suggesting that they are in a free form in blood and therefore more available to reach tissues.

  20. Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring*

    PubMed Central

    Li, He; Grigoryan, Hasmik; Funk, William E.; Lu, Sixin Samantha; Rose, Sherri; Williams, Evan R.; Rappaport, Stephen M.

    2011-01-01

    A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. PMID:21193536

  1. Stereoselective binding of chiral drugs to plasma proteins

    PubMed Central

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed. PMID:23852086

  2. Single-chain Variable Fragment Albumin Fusions Bind the Neonatal Fc Receptor (FcRn) in a Species-dependent Manner

    PubMed Central

    Andersen, Jan Terje; Cameron, Jason; Plumridge, Andrew; Evans, Leslie; Sleep, Darrell; Sandlie, Inger

    2013-01-01

    Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals. PMID:23818524

  3. A molecular dynamics approach to ligand-receptor interaction in the aspirin-human serum albumin complex.

    PubMed

    Alvarez, H Ariel; McCarthy, Andrés N; Grigera, J Raúl

    2012-01-01

    In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C(9)H(8)O(4)) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called "viscous work," that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods.

  4. A Molecular Dynamics Approach to Ligand-Receptor Interaction in the Aspirin-Human Serum Albumin Complex

    PubMed Central

    Alvarez, H. Ariel; McCarthy, Andrés N.; Grigera, J. Raúl

    2012-01-01

    In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C9H8O4) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called “viscous work,” that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods. PMID:23251150

  5. Exploring the interaction of bisphenol-S with serum albumins: a better or worse alternative for bisphenol a?

    PubMed

    Mathew, Manjumol; Sreedhanya, S; Manoj, P; Aravindakumar, C T; Aravind, Usha K

    2014-04-10

    The interaction of bisphenol-S (BPS) with serum albumins using steady-state, synchronous, time-resolved, and circular dichroism spectroscopies has been investigated. The binding interactions have also been investigated in the case of bisphenol A (BPA). The fluorescence quenching pathways are different for both of these endocrine disrupting compounds. Steady-state and time-resolved studies reveal static quenching at lower concentrations of BPS and dynamic quenching at higher concentrations. CD results also maintained the concentration dependent variation with a complete distortion of α-helices at 10(-5) M BPS. Besides this, addition of sodium dodecyl sulfate (SDS) results in the further unfolding of protein in the case of BPS, whereas time-resolved studies indicated refolding for BPA denatured human serum albumin (HSA). The entire study indicates an irreversible binding of BPS with HSA. Hence, these results reveal the possible involvement of BPS in the physiological pathway raising a health threat as already their presences in body fluids are known.

  6. Development of enzyme-linked immunosorbent assay for estimation of urinary albumin.

    PubMed

    Shrivastav, Tulsidas G; Kariya, Kiran P; Prasad, Pramod K V; Chaube, Shail K; Kumar, Dinesh

    2014-01-01

    Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13-100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38-10.32 % and 4.22-11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.

  7. Photocopy, ASBF photograph, December 1943. (HSA, Admiral Furlong Collection ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Photocopy, ASBF photograph, December 1943. (HSA, Admiral Furlong Collection - Neg. HC 03, 014). Aerial oblique of Waipio Peninsula, central part, showing sugarcane fields - U.S. Naval Base, Pearl Harbor, Waipio Peninsula, Waipo Peninsula, Pearl City, Honolulu County, HI

  8. Long-Acting Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Children

    PubMed Central

    Chambost, Hervé; Male, Christoph; Lambert, Thierry; Halimeh, Susan; Chernova, Tatiana; Mancuso, Maria Elisa; Curtin, Julie; Voigt, Christine; Li, Yanyan; Jacobs, Iris; Santagostino, Elena

    2016-01-01

    Summary A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2% of bleeding episodes were successfully treated with one or two injections of rIX-FP (95% CI: 92% to 99%), 88.7% with one injection, and 96% of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531) PMID:27583313

  9. Imidazolium ionic liquids as solvents of pharmaceuticals: influence on HSA binding and partition coefficient of nimesulide.

    PubMed

    Azevedo, Ana M O; Ribeiro, Diogo M G; Pinto, Paula C A G; Lúcio, Marlene; Reis, Salette; Saraiva, M Lúcia M F S

    2013-02-25

    In this work, the influence of imidazolium ionic liquids (ILs) on bio-chemical parameters that influence the in vivo behavior of nimesulide was evaluated. In this context, the binding of nimesulide to human serum albumin (HSA), in IL media, was studied. In parallel, the evaluation of the interaction of drug-IL systems, with micelles of hexadecylphosphocholine (HDPC), enabled the calculation of partition coefficients (K(p)). Both assays were performed in buffered media in the absence and in the presence of emim [BF(4)], emim [Ms] and emim [TfMs] 1%. Even though there was an increase of the dissociation constant (K(d)) in IL media, nimesulide still binds to HSA by means of strong interactions. The thermodynamic analysis indicates that the interaction is spontaneous for all the tested systems. Moreover, the studied systems exhibited properties that are favorable to the interaction of the drug with biological membranes, with K(p) values 2.5-3.5 higher than in aqueous environment. The studied nimesulide-IL systems presented promising characteristics regarding the absorption and distribution of the drug in vivo, so that the studied solvents seem to be good options for drug delivery. PMID:23287776

  10. Development of human serum albumin conjugated with near-infrared dye for photoacoustic tumor imaging

    NASA Astrophysics Data System (ADS)

    Kanazaki, Kengo; Sano, Kohei; Makino, Akira; Takahashi, Atsushi; Deguchi, Jun; Ohashi, Manami; Temma, Takashi; Ono, Masahiro; Saji, Hideo

    2014-09-01

    Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2=0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.

  11. Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin

    NASA Astrophysics Data System (ADS)

    Lin, Shan-Yang; Li, Mei-Jane; Wei, Yen-Shan

    2004-11-01

    We determined the secondary structure of solid-state native human serum albumin (HSA) and its precipitates induced by ethanol, captopril, or a captopril/ethanol mixture. A transmission Fourier transform infrared (FT-IR) microspectroscopy equipped with a thermal analyzer was used. The secondary structural composition of solid-state native HSA was 54% α-helices (1655 cm -1), 22% β-turns (1679 cm -1), and 23% β-sheets (1633 cm -1). After ethanol treatment, a new peak was observed at 1690 cm -1, and the peak at 1633 cm -1 was more apparent in the HSA precipitates. The corresponding compositions consisted of 59% α-helices, 17% β-turns, and 24% β-sheets. After treatment with captopril with or without ethanol, the percentage of α-helices and β-turns decreased in both HSA precipitates, but the percentage of β-sheets increased. The temperature-dependent structural transformation from α-helices/random coils to β-sheets for the solid-state HSA samples occurred at markedly different onset temperatures. The onset temperature for native HSA was 85 °C, and that for HSA precipitates obtained from ethanol, captopril, or captopril/ethanol was 100, 48 or 57 °C, respectively. The thermal-induced structural transformation from α-helices/random coils to β-sheets implies a partial unfolding structure in these HSA samples.

  12. Anti-enrofloxacin antibody production by using enrofloxacin-screened HSA as an immunogen

    NASA Astrophysics Data System (ADS)

    Liu, Chune; Lin, Hong; Cao, Limin; Jiang, Jie

    2005-07-01

    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1:250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  13. Heme-based catalytic properties of human serum albumin

    PubMed Central

    Ascenzi, P; di Masi, A; Fanali, G; Fasano, M

    2015-01-01

    Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted. PMID:27551458

  14. Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins.

    PubMed

    Sandhya, B; Hegde, Ashwini H; Seetharamappa, J

    2013-05-01

    The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%.

  15. Three-month variation of plasma pentraxin 3 compared with C-reactive protein, albumin and homocysteine levels in haemodialysis patients

    PubMed Central

    Sjöberg, Bodil; Snaedal, Sunna; Stenvinkel, Peter; Qureshi, Abdul Rashid; Heimbürger, Olof; Bárány, Peter

    2014-01-01

    Background Inflammatory markers vary considerably over time in haemodialysis (HD) patients, yet the variability is poorly defined. The aim of the study was to assess changes of plasma levels of pentraxin-3 (PTX-3), C-reactive protein (CRP), albumin and homocysteine (Hcy) over 3 months and the association between the changes in these biomarkers and mortality. Methods In 188 prevalent HD patients, inflammatory markers were measured at inclusion and after 3 months. Mortality was recorded during a median follow-up of 41 months. The changes of the biomarker levels were categorized according to change in tertile for the specific biomarker. The variation was calculated as the intra-class correlation (ICC). Mortality was analysed by Kaplan–Meier and Cox proportional hazards model. The predictive strength was calculated for single measurements and for the variation of each inflammatory marker. Results The intra-individual variation (low ICC) was largest for PTX-3 [ICC 0.44; 95% confidence interval (CI): 0.33–0.55], albumin (ICC 0.58; 95% CI: 0.49–0.67) and CRP (ICC 0.59; 95% CI: 0.51–0.68) and lowest for Hcy (ICC 0.81; 95% CI: 0.77–0.86). During follow-up, 88 patients died. Conclusions PTX-3 measurements are less stable and show higher variation within patients than CRP, albumin and Hcy. Persistently elevated PTX-3 levels are associated with high mortality. Moreover, in multivariate logistic regression we found that stable high PTX-3 adds to the mortality risk, even after inclusion of clinical factors and the three other biomarkers. The associations of decreasing albumin levels as well as low Hcy levels with worse outcome reflect protein-energy wasting. PMID:25852911

  16. Alternatives to freeze-drying for the removal of ethanol from plasma proteins. II. Gel filtration of albumin.

    PubMed

    Dickson, A J; Smith, J K

    1975-01-01

    Removal of ethanol from highly concentrated solutions of human albumin (Cohn fraction V) by gel filtration on Sephadex G-25 is hindered by the contraction of the gel in ethanolic solution, by incomplete retardation of ethanol compared with other low MW solutes, and by restricted diffusion of ethanol from the albumin zone. Despite these obstacles, the hourly capacity of such gel filtration columns, for approximately 100-fold reduction of ethanol concentration, may exceed 0.06 kg albumin per litre of column volume. The gel can be used safely at 5 degrees C for several years. The ethanol content of the final product is higher than that achieved by vacuum distillation, and it may be desirable to operate the two techniques sequentially.

  17. Capillary electrophoresis/frontal analysis versus equilibrium dialysis in dexamethasone sodium phosphate-serum albumin binding studies.

    PubMed

    Gonciarz, Anna; Kus, Kamil; Szafarz, Małgorzata; Walczak, Maria; Zakrzewska, Agnieszka; Szymura-Oleksiak, Joanna

    2012-11-01

    Plasma protein binding of drugs may have significant effect on its pharmacodynamic, toxicological and pharmacokinetic properties, since only the free drug can pass across biological membrane and get to its specific site of action. Many drugs show a high affinity to albumin which is the most abundant plasma protein. In the present study capillary electrophoresis in the frontal analysis mode (CE/FA), as promising technique for assessment of drug-protein interaction was used. The free drug concentration was measured from height of the frontal peak and calculated based on the external drug standard in absence of protein. With a known concentration of total drug, the percentage of protein bound drug was determined. The binding parameters were also estimated based on the equilibrium dialysis experiment which is considered to be a reference method. This study was designed to examine the interaction of dexamethasone sodium phosphate (DXM) with BSA and HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate buffer, I = 0.17). Using fixed, at physiological level, HSA and BSA concentrations and increasing DXM concentrations, the number of binding sites (n) and binding constant (K(a) ) was calculated from both nonlinear regression fitting and Scatchard Plot. Despite some differences, it can be concluded that the CE/FA is comparable with equilibrium dialysis, but since the first one offers advantages such as low sample consumption, short analysis time, and high separation efficiency, it can be used in high-throughput screening of drug protein binding at the early stage of drug discovery. Interspecies differences in binding of a drug to albumins have been observed and it should be taken into account in interpretation of the results.

  18. Raman, SERS, and induced circular dichroism techniques as a probe of pharmaceuticals in their interactions with the human serum albumin and p-glycoprotein

    NASA Astrophysics Data System (ADS)

    Fleury, Fabrice; Ianoul, Anatoli I.; Baggetto, Loris; Jardillier, Jean-Claude; Alix, Alain J.; Nabiev, Igor R.

    1999-04-01

    Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.

  19. Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

    PubMed Central

    Juárez, Josué; Taboada, Pablo; Mosquera, Víctor

    2009-01-01

    The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils

  20. Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408-423 in bodily fluids.

    PubMed

    Mohr, Katharina B; Zirafi, Onofrio; Hennies, Mark; Wiese, Sebastian; Kirchhoff, Frank; Münch, Jan

    2015-05-01

    Urinary levels of human serum albumin (hSA) fragment 408-423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408-423). The sandwich ELISA has a detection limit of 0.5ng/ml and is highly specific for hSA(408-423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408-423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders.

  1. Energetic domains and conformational analysis of human serum albumin upon co-incubation with sodium benzoate and glucose.

    PubMed

    Taghavi, F; Moosavi-Movahedi, A A; Bohlooli, M; Habibi-Rezaei, M; Hadi Alijanvand, H; Amanlou, M; Sheibani, N; Saboury, A A; Ahmad, F

    2014-01-01

    Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemi