Interaction Of Human Serum Albumin Research Articles

Molecular docking and spectroscopic methods to probe the interaction of acetylgliotoxin and HSA

The interaction between acetylgliotoxin and human serum albumin (HSA) has been studied by spectroscopic method. Acetylgliotoxin quenches the intrinsic fluorescence of HSA in a static quenching procedure. The binding of acetylgliotoxin to HSA causes a slight conformational change of HSA. The binding constants at different temperatures as well as enthalpy change and entropy change, are calculated according to relevant fluorescent data and Van’t Hoff equation. The hydrogen bond is a predominant intermolecular force for stabilizing the complex, which is in conformity with the results of molecular modeling study.

Kinetic and thermodynamic analysis of 9‐nitrocamptothecin hydrolysis at physiological pH in the presence and absence of human serum albumin

AbstractKinetic and thermodynamic analysis of the 9‐nitrocamptothecin (9NC) hydrolysis reaction in the presence and absence of human serum albumin (HSA) in phosphate‐buffered saline (PBS) of pH 7.4 was carried out by first derivative absorption spectroscopy. The thermodynamic parameters determined in these studies provided a mechanistic explanation toward the endothermic but yet thermodynamically favorable hydrolysis of 9NC at physiological temperature and pH. In the presence of HSA, the apparent rate constant of 9NC hydrolysis was 3–3.5 times higher than in 9NC solutions alone, whereas the apparent equilibrium constant of 9NC hydrolysis was found to increase at a higher extent in the presence of HSA than in PBS with increasing temperature, reaching almost complete hydrolysis of the 9NC to the 9NC‐carboxylate at 315.15 K. Importantly, the Ea of the 9NC hydrolysis reaction in the presence of HSA was determined to be on average 17 kJ mol−1 lower than the Ea determined in plain PBS. Moreover, analysis of binding isotherms constructed for the HSA interaction with 9NC, using infinitely cooperative and independent binding models, demonstrated an incredibly higher binding constant for the 9NC‐carboxylate form as compared to the very weak and concentration‐dependent binding for the 9NC‐lactone species at 310.15 K. Taken together, the preferential association of the carboxylate form with HSA and the lower Ea of 9NC hydrolysis in the presence of HSA provide a mechanistic explanation for the shift of lactone–carboxylate equilibria toward the carboxylate product under physiological conditions of pH and ionic strength. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 693–703, 2010

Behavior of human serum albumin on strong cation exchange resins: I. Experimental analysis

Experiments with human serum albumin on the strong cation exchange resin Fractogel EMD SE Hicap (M) were carried out. Even though human serum albumin was used at high purity, two peaks in gradient elution experiments occurred. The obtained data can be explained by considering that human serum albumin binds to Fractogel EMD SE Hicap (M) in two different binding conformations: the protein adsorbs instantaneously in the first conformation and then changes into the second one with a kinetic limitation. The two-peak behavior of human serum albumin was analyzed in detail, especially at various gradient lengths, concentrations and temperatures. Breakthrough curves were performed at four modifier concentrations and three velocities. The characteristic adsorption behavior, found for gradient experiments, was confirmed by the breakthrough curves. The two-peak elution pattern of human serum albumin was also found for other strong cation exchange resins, but not for weak cation exchange resins. It is concluded that the described behavior is peculiar for the interaction of human serum albumin with the strong cation exchange ligand of the resin.

Modeling the Time Evolution of the Nanoparticle-Protein Corona in a Body Fluid

BackgroundNanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona.ResultsWe developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL.ConclusionsThe simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine.

Bovine and Human Serum Albumin Interactions with 3-Carboxyphenoxathiin Studied by Fluorescence and Circular Dichroism Spectroscopy

The interactions of 3-carboxyphenoxathiin with Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) have been studied by fluorescence and circular dichroism spectroscopy. The binding of 3-carboxyphenoxathiin quenches the BSA and HSA fluorescence, revealing a 1:1 interaction with a binding constant of about 105 M-1. In addition, according to the synchronous fluorescence spectra of BSA and HSA in presence of 3-carboxyphenoxathiin, the tryptophan residues of the proteins are most perturbed by the binding process. Finally, the distance between the acceptor, 3-carboxyphenoxathiin, and the donor, BSA or HSA, was estimated on the basis of the Förster resonance energy transfer (FRET). The fluorescence results are correlated with those obtained from the circular dichroism spectra, which reveal the change of the albumin conformation during the interaction process.

Investigation of the interaction between endocrine disruptor bisphenol A and human serum albumin

In this study, the interaction of the endocrine disruptor bisphenol A (BPA) and human serum albumin (HSA) was investigated by molecular modelling, fluorescence, ultraviolet–visible spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD). The association constants between BPA and HSA were determined using the Scatchard equation. The thermodynamic parameters of the binding reaction (Δ G 0, Δ H 0 and Δ S 0) were measured, and they indicated the presence of hydrophobic forces in the BPA–HSA interaction, which agreed well with the results from molecular modelling. The alterations of protein secondary structure in the presence of BPA were confirmed by UV–vis, FT-IR and CD spectroscopy. Lastly, the average binding distance, r, between BPA and HSA was evaluated and found to be 1.82 nm according to Förster’s theory of non-radiation energy transfer.

Complexes between Fluorescent Cholic Acid Derivatives and Human Serum Albumin. A Photophysical Approach To Investigate the Binding Behavior

Interaction between bile acids and plasma proteins has attracted considerable attention over past decades. In fact, binding of bile acids to human serum albumin (HSA) determines their level in plasma, a value that can be used as a test for liver function. However, very little is known about the role that bile acids-HSA complexes play in hepatic uptake. In the present paper, we report on the utility of the singlet excited state properties of 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent derivatives of cholic acid (ChA); namely, 3alpha-NBD-ChA, 3beta-NBD-ChA, 3beta-NBD-ChTau, 7alpha-NBD-ChA, and 7beta-NBD-ChA to clarify key aspects of bile acids-HSA interactions that remain poorly understood. On the basis of either absorption or emission measurements, formation of NBD-ChA@HSA complexes with 1:1 stoichiometry has been proven. Enhancement of the fluorescence emission upon addition of HSA has been used for determination of the binding constants, which are in the range of 10(4) M(-1). Energy transfer from tryptophan to NBD-ChA occurs by a FRET mechanism; the donor-acceptor distances have been determined according to Forster's theory. The estimated values (27-30 A) are compatible with both site I and site II occupancy and do not provide sufficient information for a safe assignment; however, fluorescence titration using warfarin (site I probe) and ibuprofen (site II probe) for displacement clearly indicates that the employed cholic acid derivatives bind to HSA at site I.

A Thermodynamic Study on the Binding of Human Serum Albumin with Lanthanum Ion

AbstractThermodynamics of the interaction between lanthanum(III) ion, La3+, and human serum albumin (HSA), was investigated at pH 7.0 and 300 K in Tris‐HCl buffer by isothermal titration calorimetry. A solvation model was used to reproduce the enthalpies of HSA interaction with La3+. The solvation parameters recovered from our new model were attributed to the structural change of HSA and its biological activity. The interaction of HSA with La3+ showed a set of two binding sites with negative cooperativity.

Interactions of human serum albumin with meloxicam: Characterization of binding site

Human serum albumin (HSA) is the most prominent protein in plasma. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme, bilirubin and drugs. Here, we report the effect of new generation, non-steroidal anti-inflammatory drug (NSAID) meloxicam on the albumin conformation and ligand binding. In the present work the interaction of meloxicam with HSA in aqueous solution at physiological pH has been investigated through circular dichroism and fluorescence spectroscopy. The strong quenching of the fluorescence clearly indicated that the binding of the drug to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. This was confirmed by the destabilization of the warfarin binding site. CD and fluorescence spectroscopic results showed marked reductions (about 40% decrease in the CD Cotton effect intensity, and ∼15% decrease of the fluorescence intensity) in the affinity of albumin for bilirubin upon meloxicam binding. The strong inhibition of warfarin and ANS bound to protein after meloxicam modification compared with aspirin confirms that the binding site of both drugs is not the same.

Interactions of Human Serum Albumin with Phenothiazine Drugs: Insights from Fluorescence Spectroscopic Studies

AbstractThis study was based on the use of fluorescence quenching and differentiation between static and dynamic quenching, as well as energy transfer for distance measurements. The interactions of human serum albumin with phenothiazine drugs, i.e. phenothiazine, chlorpromazine, perphenazine and promethazine, were studied and extrapolated important information on quenching mechanism, types of interaction force, binding‐site number and distance between Trp214 in HSA and the bound drugs. This study has great significance in methodology and understanding the protein‐drug interaction mechanism.

The effect of methylamine on the solution structures of human and bovine serum albumins

Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions including transporting a variety of compounds. Methylamine, a monoamine with one positive charge complexes with protein and alters protein secondary structure. The aim of this study was to examine the interactions of human serum albumin (HSA) and bovine serum albumin (BSA) with methylamine at physiological conditions, using constant protein concentration and various monoamine concentrations. FTIR, UV–vis, CD and fluorescence spectroscopic methods were used to analyse methylamine binding mode, the binding constant and the effects of monoamine on HSA and BSA stability and conformations. Structural analysis showed that methylamine binds HSA and BSA via hydrophilic (polypeptide and amine polar groups) and hydrophobic interactions with overall binding constants of K met-HSA = 2.42 (±0.5) × 10 2 M −1 and K met-BSA = 1.34 (±0.3) × 10 3 M −1 with the number of bound methylamine around one molecule per protein. Methylamine complexation alters protein conformation by major reduction of α-helix and increase in random coil and turn structures indicating a partial protein unfolding.

Dendrimers Bind Human Serum Albumin

Dendrimers are synthetic, highly branched, spherical macromolecules with nanometer dimensions and potential applications in DNA and drug delivery systems. Human serum albumin (HSA) is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interaction of human serum albumin with several dendrimers such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant protein concentration and various dendrimer compositions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to analyze macromolecule binding mode, the binding constant and the effects of dendrimers complexation on HSA stability and conformation. Structural analysis showed that dendrimers bind HSA via polypeptide polar groups (hydrophilic) with number of bound polymer (n) 1.08 (mPEG-PAMAM-G3), 1.50 (mPEG-PAMAM-G4), and 0.96 (PAMAM-G4). The overall binding constants estimated were of KmPEG-G3=1.3 (+/-0.2)x10(4) M(-1), KmPEG-G4=2.2 (+/-0.4)x10(4) M(-1), and KPAMAM-G4=2.6 (+/-0.5)x10(4) M(-1). HSA conformation was altered by dendrimers with a major reduction of alpha-helix and increase in random coil and turn structures suggesting a partial protein unfolding.

Study on Binding of Drug to Serum Protein

After being distributed in the circulating blood, drugs bind to serum proteins varying degrees. In general, such binding is reversible, and a dynamic equilibrium exists between the bound and unbound molecular species. It is believed that unless there is a specific transport system (e.g. receptor-mediated endocytosis, protein-mediated transport), only unbound drugs are able to penetrate through biomembranes, are distributed to tissues, and undergo metabolism and glomerular filtration. It is also believed that only unbound molecules present in target tissues can exert their pharmacological effects, and that the concentration of unbound molecules in tissues is in proportion to the drug serum concentration. Therefore, drug-serum protein binding is critically involved in the manifestation of the pharmacological effects of a drug as well as its pharmacokinetics. Among serum proteins, human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) play important roles in protein binding for many drugs, which is of key importance to drug distribution in the body. In addition, they are widely used in clinical settings as blood preparations and drug delivery system carriers. It is thus of great importance from the viewpoint of pharmaceutical science to clarify the structure, function, and pharmaceutical properties of HSA and AGP. Accordingly, since starting my laboratory, the focus of my research has involved molecular pharmaceutical studies on the interactions of drugs and HSA and AGP for the purpose of applying these findings to clinical fields, such as drug treatment, diagnosis and drug discovery. In this review, the molecular properties of HSA and AGP will be briefly outlined. The static and dynamic topology of drug binding sites on these proteins, investigated by various spectroscopic techniques, X-ray crystallography, quantitative structure-activity relationships, molecular modeling, photo affinity labeling, site-directed mutagenesis etc., changes in the serum protein binding of drugs in pathological conditions, such as liver and kidney failure and various inflammation diseases and factors contributing to the changes will then be summarized. Finally, cases in which protein binding displacement can be applied to medical fields will also be introduced.

A high performance method for thermodynamic study on the binding of human serum albumin with erbium chloride

Abstract Thermodynamics of the interaction between erbium(III) chloride, Er3+, with human serum albumin (HSA), was investigated at pH 7.0 and in phosphate buffer by isothermal titration calorimetry. Our recently, solvation model was used to reproduce the enthalpies of HSA interaction by Er3+ over a broad range of metal ion concentration. The solvation parameters recovered from our new model, attributed to the structural change of HSA and its biological activity. The binding parameters for the interaction of Er3+ and HSA indicate that the concentrations of Er3+ have no significant effects on the structure of HSA.

Structural Analysis of Human Serum Albumin Complexes with Cationic Lipids

Human serum albumin (HSA) is a major transporter for delivering several endogenous compounds including fatty acids in vivo. Even though HSA is the primary target of fatty acid binding, the effects of cationic lipid on protein stability and conformation have not been investigated. The aim of this study was to examine the interaction of human serum albumin (HSA) with helper lipids--cholesterol (Chol) and dioleoylphosphatidylethanolamine (DOPE)--and with cationic lipids--dioctadecyldimethylammonium bromide (DDAB) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), at physiological conditions, using constant protein concentration and various lipid contents. Fourier transform infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopic methods were used to analyze the lipid binding mode, the binding constant, and the effects of lipid interaction on HSA stability and conformation. Structural analysis showed that cholesterol and DOPE (helper lipids) interact mainly with HSA polypeptide polar groups and via hydrophobic moieties. Hydrophobic interactions dominate the binding of cationic lipids to HSA. The number of bound lipids (n) calculated was 1.22 (cholesterol), 1.82 (DDAB), 1.76 (DOPE), and 1.56 (DOTAP). The overall binding constants estimated were KChol=2.3 (+/-0.50)x10(3) M(-1), KDDAB=8.9 (+/-0.95)x10(3) M(-1), KDOTAP=9.1 (+/-0.90)x10(3) M(-1), and KDOPE=4.7 (+/-0.70)x10(3) M(-1). HSA conformation was stabilized by cholesterol and DOPE with a slight increase of protein alpha-helical structures, while DOTAP and DDAB induced an important (alpha-->beta) transition, suggesting a partial protein unfolding.

Interaction of human serum albumin with charge transfer probeethyl ester of N,N-dimethylamino naphthyl acrylic acid: An extrinsic fluorescence probe for studying protein micro-environment

The charge transfer (CT) probe ethyl ester of N,N-dimethylamino naphthyl acrylic acid (EDMANA) bound to Human Serum Albumin (HSA) serves as an efficient reporter of the polarity and conformational changes of protein in aqueous buffer (Tris-HCl buffer, pH=7.03) and in presence of denaturant, quencher and reverse micelles. The change in fluorescence intensity and the position of emission maxima of EDMANA in presence of HSA well reflect the nature of binding and location of the probe inside the proteinous environment. The increase in steady state anisotropy values with increase of protein concentration indicate restriction imposed on the mobility of the probe molecules in the proteinous medium. The results of fluorescence quenching of EDMANA by acrylamide, Fluorescence Resonance Energy Transfer (FRET) and Red Edge Excitation Shift (REES) studies throw light on the accessibility to the probe bound to HSA and hence indicate the probable location of the probe within the hydrophobic cavity of HSA. The complicated nature of protein unfolding in presence of urea is well studied by change in the fluorescence properties of EDMANA bound to HSA protein.

Saturation transfer difference nuclear magnetic resonance study on the specific binding of ligand to protein

Ligand-based nuclear magnetic resonance (NMR) approaches have shown great promise in the study of ligand–protein interaction. But these approaches suffer from interference from the nonspecific binding. Here a saturation transfer difference (STD) NMR method to map the group epitope and to measure the dissociation constant ( K D) of specific interaction between ligand and protein is presented. The interference from nonspecific binding was corrected by recording STD NMR spectra of ligand–protein solutions with and without inhibitor saturating the mutually specific binding site and subtracting one from the other. The method was examined with l-tryptophan (Trp), naproxen (Nap), and human serum albumin (HSA) as model ligand, inhibitor, and protein, respectively. Results agree well with other reports of Trp–HSA interaction.

Human serum albumin interaction with oxaliplatin studied by capillary isoelectric focusing with the whole column imaging detection and spectroscopic method

Capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID) was used to investigate drug-protein interactions. This study was designed to examine the interaction between the platinum-based anticancer drug, oxaliplatin, with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 10 to 100 μM and a constant concentration of HSA (5.0 × 10 −5 M). The reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48 and 72 h at 37 °C in a water bath. The CIEF results indicate that with increasing the drug concentration, the complex formation of protein adducts increased compared to low-drug concentrations and major structural changes were observed as the incubation time progressed. The altered CIEF profile demonstrated the possible conformation change due to the binding of the drug. Results also showed a significant protein's p I shift for higher HSA–oxaliplatin incubation ratios. Furthermore, spectroscopic evidence shows that oxaliplatin caused the fluorescence quenching of HSA by formation of HSA–oxaliplatin complex. Using the Stern–Volmer equation, the quenching constants were calculated in the linear range. The quenching rate constants K q at three different temperatures indicating the presence of static quenching mechanism in the interactions of oxaliplatin with HSA. This paper describes the validity of the CIEF-WCID technique for the study of protein–drug interactions and provides useful information and insight into the interaction of anticancer drugs with HSA.

Spectroscopic Studies on the Comparative Interaction of Cationic Single-Chain and Gemini Surfactants with Human Serum Albumin

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.

Interaction of anthracycline disaccharide with human serum albumin: Investigation by fluorescence spectroscopic technique and modeling studies

Anthracyclines are considered to be some of the most effective anticancer drugs for cancer therapy. However, drug resistance and cardiotoxicity of anthracyclines limit their clinical application. An 3′-azido disaccharide analogue of daunorubicin, 7-[4- O-(2,6-dideoxy-3- O-methyl-α- l- arabino-hexopyranosyl)-3-azido-2,3,6-trideoxy-α- l- lyxo-hexopyranosyl]daunorubicinone (ADNR-3), was shown to exhibit 10-fold better activity than parent compound daunorubicin against the drug-resistant cells and completely overcomes the drug resistance with same IC 50 in both drug-resistant and drug-sensitive cells. In this paper, the interactions between ADNR-3 and human serum albumin (HSA) have been studied by spectroscopic techniques. By the analysis of fluorescence spectrum and fluorescence intensity, it was observed that the ADNR-3 has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The association constants of ADNR-3 with HSA were determined at different temperatures based on fluorescence quenching results. The negative Δ H and positive Δ S values in case of ADNR-3–HSA complexes showed that both hydrogen bonds and hydrophobic interactions play a role in the binding of ADNR-3 to HSA. Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between ADNR-3 and HSA changed the molecular conformation of HSA and the hydrophobic interactions play a major role in ADNR-3–HSA association. Moreover, the study of computational modeling indicated that ADNR-3 could bind to the site I of HSA and hydrophobic interaction was the major acting force for the second type of binding site, which was in agreement with the thermodynamic analysis. The distance, r, between donor (HSA) and acceptor (ADNR-3) was obtained according to the Förster's theory of non-radiation energy transfer. In addition, the effects of common ions on the binding constants of ADNR-3–HSA complexes were also investigated.