Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

Comparative studies of unfolding and binding of ligands to human serum albumin in the presence of fatty acid: spectroscopic approach

This study was undertaken to investigate the influence of fatty acid binding on the unfolding of HSA and how the fatty acid molecules can influence and/or compete with other ligand molecules bound to the protein. The equilibrium unfolding of fatted and fatty acid free HSA was measured by overlapping of unfolding transition curves monitored by different probes for secondary and tertiary structure and determining changes in free energy of unfolding. Proteins stability was studied by fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism techniques. We have suggested a "molten globule" like intermediate state of HSA at a fairly high concentration of GnHCl (3.2 for fatty acid free and 3.6 for fatted). The free energy of stabilization (DeltaG(D)(H2O)) in the presence of fatty acid was found to be 900 cal mol(-1). We also analyze the effects of fatty acid on binding of ligands using spectroscopic technique and reported the equilibrium constants and free energies obtained from the binding and unfolding experiments.     

Interaction of glucose oxidase with ionic surfactants: a microcalorimetric study

The enthalpies of interaction of glucose oxidase at 25°C with a homologous series of n-alkyltrimethylammonium bromides (TABs) at pH 10 and a homologous series of n-alkylsulfates at pH 3.2 have been measured by microcalorimetry. For the n-dodecyl member of each series, DTAB and sodium n-dodecylsulfate (SDS), the binding of the surfactants to glucose oxidase as measured by equilibrium dialysis has been used in combination with the enthalpy data to obtain the Gibbs energy ( ), enthalpy ( ) and entropy ( ) of binding per surfactant molecule as a function of the number of surfactant molecules bound ( ). The thermodynamic parameters for the glucose oxidase interaction with DTAB at pH 10 and SDS at pH 3.2 are very similar and show that the interactions are entropically driven. The observed enthalpies of interaction of glucose oxidase with the homologous n-alkylsulfates have been analysed in terms of the interactions between the anionic surfactant head group and cationic sites on the protein, hydrophobic binding and the thermal contributions arising from protein unfolding. At surfactant concentrations of 0.5 c.m.c., the enthalpy of unfolding of glucose oxidase is estimated to be 3610 ± 560 kJ mol⁻¹.     

Mechanistic investigation of domain specific unfolding of human serum albumin and the effect of sucrose

This study is devoted to understand the unfolding mechanism of a multidomain protein, human serum albumin (HSA), in absence and presence of the sucrose by steady‐state and time‐resolved fluorescence spectroscopy with domain specific marker molecules and is further being substantiated by molecular dynamics (MD) simulation. In water, the domain III of HSA found to unfold first followed by domains I and II as the concentration of GnHCl is increased in the medium. The sequential unfolding behavior of different domains of HSA remains same in presence of sucrose; however, a higher GnHCl concentration is required for unfolding, suggesting stabilizing effect of sucrose on HSA. Domain I is found to be most stabilized by sucrose. The stabilization of domain II is somewhat similar to domain I, but the effect of sucrose on domain III is found to be very small. MD simulation also predicted a similar behavior of sucrose on HSA. The stabilizing effect of sucrose is explained in terms of the entrapment of water molecules in between HSA surface and sucrose layer as well as direct interaction between HSA and sucrose.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.     

Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism

The interfacial behavior of mixed human serum albumin (HSA)/sodium perfluorooctanoate (C8FONa) solutions is examined by using two experimental techniques, pendant drop tensiometry and circular dichroism spectroscopy. Through the analysis of the surface tension of the mixed solutions, surface competitive adsorption at the air-water interface between C8FONa and HSA is detected. The dynamic adsorption curves exhibit the distinct regimes in their time-dependent surface tension. The nature of these regimes is further analyzed in terms of the variation of the molecules surface areas. As a consequence, a compact and dense structure was formed where protein molecules were interconnected and overlapped. Thus, a reduction of the area occupied per molecule from 100 to 0.2 nm(2) is interpreted as a gel-like structure at the surface. The presence of the surfactant seems to favor the formation of this interfacial structure. Finally, measurements of circular dichroism suggests a compaction of the protein due to the association with the surfactant given by an increase of alpha-helix structure in the complexes as compared to that of pure protein.     

Interaction of taxol with human serum albumin

Taxol (paclitaxel) is an anticancer drug, which interacts with microtuble proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures (Nogales et al., Nature 375 (1995) 424-427). This study was designed to examine the interaction of taxol with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 0.0001-0.1 mM, and HSA (fatty acid free) concentration of 2% w/v. Gel electrophoresis, absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second-derivative resolution enhancement were used to determine the drug binding mode, binding constant and the protein secondary structure in the presence of taxol in aqueous solution. Spectroscopic evidence showed that taxol-protein interaction results into two types of drug-HSA complexes with overall binding constant of K=1.43 x 10(4) M(-1). The molar ratios of complexes were of taxol/HSA 30/1 (30 mM taxol) and 90/1 (90 mM taxol) with the complex ratios of 1.9 and 3.4 drug molecules per HSA molecule, respectively. The taxol binding results in major protein secondary structural changes from that of the alpha-helix 55 to 45% and beta-sheet 22 to 26%, beta-anti 12 to 15% and turn 11 to 16%, in the taxol-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of taxol in aqueous solution.     

Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site

Human serum albumin (HSA) is known to exist as N (pH approximately 7), B (pH approximately 9), and F (pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) accumulate in the urea induced unfolding pathway of HSA around 4.8-5.2 M urea concentrations. These states displayed characteristic structure and functions. To elucidate the ciprofloxacin (CFX) binding behavior of HSA, the binding of ciprofloxacin with these conformational states of human serum albumin (HSA) has been investigated by fluorescence spectroscopy. The binding constant (K) for N, B, F, and I conformation of HSA were 6.92 x 10(5), 3.87 x 10(5), 4.06 x 10(5), and 2.7 x 10(5) M(-1) and the number of binding sites (n) were 1.26,1.21, 1.16, and 1.19, respectively. The standard free energy changes (DeltaGbinding(0)) of interaction were found to be -33.3 (N isomer), -31.8 (B isomer), -32 (F isomer), and -30.0 kJ mol(-1) respectively. By using unfolding pathway of HSA, domain II of HSA has been assigned to possess binding site of ciprofloxacin. Plausible correlation between stability of CFX-N and CFX-B complexes and drug distribution have been discussed. At plasma concentration of HSA fraction of free CFX, which contributes potential to its rate of transport across cell membrane, was found to be approximately 80% more for B isomers compared to N isomers of HSA. The conformational changes in two physiologically important isomers of HSA (N and B isomers) upon ciprofloxacin binding were evaluated by measuring far, near-UV CD, and fluorescence properties of the CFX-HSA complex.     

Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies

In this work, fluorescence spectroscopy in combination with circular dichroism spectroscopy and molecular modeling was employed to investigate the binding of 10-hydroxycamptothecin (HCPT) to human serum albumin (HSA) under simulative physiological conditions. The experiment results showed that the fluorescence quenching of HSA by HCPT was a result of the formation of HCPT–HSA complex. The corresponding association constants (K _a) between HCPT and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The results of thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bonds and van der Waals forces played major roles for HCPT–HSA association. Site marker competitive displacement experiment indicated that the binding of HCPT to HSA primarily took place in sub-domain IIA (site I). Molecular docking study further confirmed the binding mode and the binding site obtained by fluorescence and site marker competitive experiments. The conformational investigation showed that the presence of HCPT decreased the α-helical content of HSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of HSA molecules.     

Water-in-silicone oil emulsion stabilizing surfactants formed from native albumin and alpha,omega-triethoxysilylpropyl-polydimethylsiloxane

Contact with hydrophobic silicones frequently leads to protein denaturation. However, it is demonstrated that albumin in water-in-silicone oil emulsions retains its native structure in the presence of a functional, triethoxysilyl-terminated silicone polymer, TES-PDMS. Both HSA and TES-PDMS were essential for the formation of stable water-in-silicone oil emulsions: attempts to generate stable emulsions using independently either the protein or the functionalized silicone as a surfactant failed. Confocal microscopy indicated that the human serum albumin (HSA) preferentially adsorbed at the oil/water interface, even in the presence of another protein (glucose oxidase). A variety of experiments demonstrated that the hydrolysis of the Si-OEt groups on the functional silicone occurred only to a limited extent, consistent with the absence of a covalent linkage between the silicone and protein, or of cross-linked silicones at the interface. The fluorescence spectra of HSA extracted from the emulsions, front-faced fluorescence experiments on the HSA/silicone emulsion itself, and HSA/salicylate binding studies all demonstrated that the stability of the water/oil interface decreased as the protein began to unfold: unfolding of the protein in the emulsion was slower than in aqueous solution. The experimental evidence indicated that the interaction between HSA and TES-PDMS is not associated with either homomolecular (HSA/HSA; TES-PDMS/TES-PDMS) interactions or with covalent linkage between two the polymers. Rather, the data is consistent with the direct binding of unhydrolyzed Si(OEt) 3 groups to native HSA. The nature of these interactions is discussed.     

Impact of Potential Blockers on Ru(III) Complex Binding to Human Serum Albumin

The effects of aspirin, vitamin B2 and warfarin as potential blockers of the ruthenium binding sites in HSA were investigated through UV/visible, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy ICP(AES). The studies on the interactions of several biologically relevant molecules with HSA have shown that drugs like aspirin or warfarin may strongly influence the interaction of serum protein with anticancer drugs. It can derive from the influence of the drug on protein conformation or binding close to binding site of anticancer drug. Aspirin, vitB2 and warfarin bind to IIA subdomain leading to partial blocking of the ruthenium binding site in HSA.     

In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride

The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.     

Saffron carotenoids (crocin and crocetin) binding to human serum albumin as investigated by different spectroscopic methods and molecular docking

Therapeutic effects of saffron ingredients were studied in some diseases. The pharmacokinetics and pharmacodynamics of these ingredients were also studied, but their transport mechanism is not clearly known. Serum albumin has been known as the most important transporter of many drugs in the body that affects their disposition, transportation, and bioavailability. Here, we investigated the interaction of crocin (Cro) with HSA, for the first time, and compared with the crocetin (Crt)–HSA interaction. UV and fluorescence spectroscopy, circular dichroism (CD), and molecular docking was applied to investigate the possibility and mechanism of binding of HSA with these natural carotenoids. The gradually addition of Cro increased HSA absorbency at 278 nm, while Crt decreased it. Both of these changes induced HSA unfolding that was confirmed by the decreased α-helix content, as determined by the CD. Both carotenoids quenched HSA fluorescence emission, but with different mechanisms. The Stern–Volmer plots indicated a dynamic quenching of intrinsic emission of HSA due to Cro addition, while Crt quenching followed both static and dynamic quenching mechanisms. Docking results indicated binding of Cro/Crt in sub-domain IIA, Sudlow site I of HSA, which accompanied with the hydrogen bonding of Cro/Crt with Tyr138. The interaction of these ligands (Cro/Crt) caused HSA unfolding and affects the hydrophobic environment of Trp241, which result in the quenching of Trp fluorescence. The UV spectroscopy and fluorescence quenching data indicated the differences in the mechanisms of interaction of Cro/Crt with HSA, which is due to the differences in the structure and hydrophobicity of these ligands.     

The effect of metals on SDS-induced partially folded states of CopC

In this work, the unfolding of CopC was used to elucidate details of the protein structure through different spectroscopic techniques. The interactions of CopC and its mutants with the anionic surfactant sodium dodecyl sulfate (SDS), guanidinium hydrochloride, and urea were monitored by fluorescence spectroscopy, far-UV circular dichroism spectroscopy, and fluorescence lifetime measurements. The interaction of SDS with CopC resulted in the formation of a partially folded intermediate. In this intermediate, the structure of the C-terminal is unfolded, whereas the N-terminal retains the native structure. Further, we have explored the effects of metals on the intermediate in aqueous surfactant. The results suggested that the Ag⁺ ion has a large effect on the unfolding induced by SDS. In addition, the binding capacity of the different unfolding degree protein toward Cu²⁺ indicated the high stability of the N-terminal. The protein–Cu²⁺ unfolding induced by guanidinium hydrochloride and urea caused the binding of Cu²⁺ to increase the stability of the N-terminal, which resulted in an intermediate in the unfolding process. The first transition corresponded to unfolding of the C-terminal, and the second transition was attributed to unfolding of the N-terminal. Furthermore, the anisotropy decay indicated that the motion of tryptophan occurred at a higher urea concentration, which suggested the high stability of the N-terminal. Steered molecular dynamics simulations also indicated that the structure of the N-terminal was rigid.     

Spectroscopic and molecular modeling studies on the binding of the flavonoid luteolin and human serum albumin

Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV‐Vis absorption, fluorescence, Raman and surface‐enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp‐214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of α‐helix and increase of β‐turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 917–927, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.     

Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.     

Chronic liver and renal diseases differently affect structure of human serum albumin

Human serum albumin (HSA) binding with endogenous metabolites and drugs is substantially decreased in chronic renal and liver diseases. To test the hypothesis that the decreased binding ability is caused by conformational changes of the protein, we analyzed infrared and Raman spectra of HSA isolated from healthy donors and patients with chronic uremia and liver cirrhosis. Uremia did not affect the secondary structure of HSA but modified the environment of its Asp/Glu residues. Liver cirrhosis increased the amount of extended and beta-structures, modified the environment of Asp/Glu and Tyr side chains, and changed the configuration of disulfide bridges in albumin molecules. The conformational changes of "cirrhotic" albumin were not caused by reversibly bound ligands and resembled a partial unfolding of the protein induced by adsorption on the charcoal surface. The dramatic structural alterations of HSA in liver cirrhosis may be caused by its oxidative modification and might underlie the decreased binding ability and changed body distribution of albumin.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.