Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.     

Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.     

Structural insights into human serum albumin-mediated prostaglandin catalysis

Previous studies have shown that many arachidonic acid metabolites bind to human serum albumin (HSA) and that the metabolism of these molecules is altered as a result of binding. The present study attempted to gain insights into the mechanisms by which prostaglandins bound to subdomain 2A of HSA are metabolized by catalytic processes. The breakdown of the prostaglandin 15-keto-PGE(2) to 15-keto-PGA(2) and 15-keto-PGB(2) in the presence of wild-type HSA and a number of subdomain 2A mutants was examined using a previously validated spectroscopic method which monitors absorbance at 505 nm. The species examined using this method were wild-type HSA, K195M, K199M, F211V, W214L, R218M, R218P, R218H, R222M, H242V, R257M, and bovine serum albumin. Previous studies of HSA-mediated catalysis indicated that the breakdown of HSA-bound prostaglandins results from an alkaline microenvironment in the binding site. Our results show that the catalytic breakdown of HSA-bound 15-keto-PGE(2) to 15-keto-PGB(2) results from two specific processes which are modulated by specific amino acid residues. Specifically, some amino acid residues modulate the rate of step 1, the conversion of 15-keto-PGE(2) to 15-keto-PGA(2), while other residues modulate the rate of step 2, the conversion of 15-keto-PGA(2) to 15-keto-PGB(2). Some residues modulate the rate of steps 1 and 2. In total, while our results support the involvement of certain basic amino acid residues in the catabolism of HSA-bound 15-keto-PGE(2), our data suggest that metabolism of HSA-bound prostaglandins may be a more complex and specific process than previously thought.     

Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells

Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.     

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.     

Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXα revealed by phage display

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR.     

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.     

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.     

Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.     

Histidine146 of human serum albumin plays a prominent role at the interface of subdomains IA and IIA in allosteric ligand binding

Fatty acids are endogenous ligands of human serum albumin (HSA) that induce conformational changes and participate in allosteric ligand binding to HSA. In a previous study, we showed that, when myristate (MYR) is present, the binding of [(14) C]ketoprofen (KP) to subdomain IA of HSA was increased, indicating that, when MYR binds to HSA, a new binding site in formed in that region. Meanwhile, an N-B transition has been reported to increase the binding of ligands at alkaline pH when the status of albumin is the B-conformer. Six histidine single mutants of HSA, H9A, H39A, H67A, H105A, H128A and H146A were produced and photolabeled with [(14) C]KP at pH 6.5, 7.4 and 8.2 and the role of each histidine in causing the N-B transition induced allosteric ligand binding was examined. Cyanogen bromide cleavage of the photolabeled native HSA showed that subdomain IA was the site of the allosteric binding of KP at pH 8.2. From the photolabeling results, H146 was found to play a prominent role whilst H128 played little or no role in the allosteric binding. However, the remaining 4 mutants did not show a clear photolabeling pattern that was similar to either native HSA or H146A and, as a result, no firm conclusions can be made. An additional histidine mutant, H146I, was produced to confirm the results for H146A. A similar experiment using H146I showed that a benzene ring-like structure at position 146 is required for the allosteric ligand binding to occur.     

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.     

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.     

Identification of the cleavage sites of oxidized protein that are susceptible to oxidized protein hydrolase (OPH) in the primary and tertiary structures of the protein

Amino acid sequences in H(2)O(2)-oxidized bovine serum albumin (BSA) that are susceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 22, 20, and 8 kDa) were produced as analyzed by SDS-PAGE. N-Terminal amino acid sequence analysis of these fragments indicated that oxidized BSA was cleaved by OPH at three major sites, Leu218-Ser219, Tyr410-Thr411, and Phe506-Thr507, at an early stage of the proteolytic degradation. In the three-dimensional structure of BSA deduced by computer modeling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxidation on the tertiary structure of BSA was then investigated by hypothetically replacing all the four methionine and two tryptophan residues with their oxidized forms, methionine sulfoxide and N'-formyl-kynurenine, respectively. The three-dimensional structure of the hypothetically oxidized BSA indicated that all the three cleavage sites in the protein could become more exposed to the solvent than in unoxidized BSA. These results suggest that, upon oxidation of BSA, the amino acid sequences that are potentially cleavable by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating the cleavage sites of oxidized protein by oxidized protein-selective protease, suggesting the possible mechanism of oxidized protein-selective degradation by the enzyme.     

Roles of the heme distal residues of FixL in O2 sensing: a single convergent structure of the heme moiety is relevant to the downregulation of kinase activity

FixL is a heme-based O(2) sensor, in which the autophosphorylation is regulated by the binding of exogenous ligands such as O(2) and CN(-). In this study, mutants of the heme distal Arg200, Arg208, Ile209, Ile210, and Arg214 residues of SmFixL were characterized biochemically and physicochemically, because it has been suggested that they are significant residues in ligand-linked kinase regulation. Measurements of the autoxidation rate, affinities, and kinetics of ligand binding revealed that all of the above residues are involved in stabilization of the O(2)-heme complex of FixL. However, Arg214 was found to be the only residue that is directly relevant to the ligand-dependent regulation of kinase activity. Although the wild type and R214K and R214Q mutants exhibited normal kinase regulation, R214A, R214M, R214H, and R214Y did not. (13)C and (15)N NMR analyses for (13)C(15)N(-) bound to the truncated heme domains of the Arg214 mutants indicated that, in the wild type and the foregoing two mutants, the heme moiety is present in a single conformation, but in the latter four, the conformations fluctuate possibly because of the lack of an interaction between the iron-bound ligand and residue 214. It is likely that such a rigid conformation of the ligand-bound form is important for the downregulation of histidine kinase activity. Furthermore, a comparison of the NMR data between the wild type and R214K and R214Q mutants suggests that a strong electrostatic interaction between residue 214 and the iron-bound ligand is not necessarily required for the single convergent structure and eventually for the downregulation of FixL.     

Effects of γ‐Irradiation on the Molecular Structures and Functions of Human Serum Albumin

In this paper, we use spectroscopic methods (fluorescence spectroscopy, UV absorption spectroscopy, and circular dichroism (CD) spectroscopy) to elucidate the effects of reactive oxygen species generated by γ‐irradiation on the molecular properties of human serum albumin (HSA). The results of fluorescence spectroscopy indicated that oxidation by γ‐irradiation can lead to conformational changes of HSA. Data of CD spectra suggested that with the increase of radiation dose the percentage of α‐helix in HSA has decreased. The determination of protein hydrophobicity showed that the effective hydrophobicity of HSA decreased up to 62% compared to the native HSA solution due to the exposure to the γ‐irradiation. Furthermore, small changes in the esterase‐like activity of HSA were introduced because of oxidation. The content of bityrosine increased markedly, suggesting that the oxidized HSA was aggregated. Moreover, there was no obvious change in the molecular properties of HSA with low γ‐irradiation dose. Changes happened when the irradiation dose exceeded 200 Gy.     

Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.     

Role of the 207-218 peptide region of Moloney murine leukemia virus integrase in enzyme catalysis

X-ray diffraction data on a few retroviral integrases show a flexible loop near the active site. By sequence alignment, the peptide region 207-218 of Mo-MLV IN appears to correspond to this flexible loop. In this study, residues H208, Y211, R212, Q214, S215 and S216 of Mo-MLV IN were mutated to determine their role on enzyme activity. We found that Y211A, R212A, R212K and Q214A decreased integration activity, while disintegration and 3′-processing were not significantly affected. By contrast H208A was completely inactive in all the assays. The core domain of Mo-MLV integrase was modeled and the flexibility of the region 207-216 was analyzed. Substitutions with low integration activity showed a lower flexibility than wild type integrase. We propose that the peptide region 207-216 is a flexible loop and that H208, Y211, R212 and Q214 of this loop are involved in the correct assembly of the DNA-integrase complex during integration.     

Inhibition of the peroxidation of linoleic acid by the flavonoid quercetin within their complex with human serum albumin

This work provides a quantitative kinetic analysis of oxidative pathways involving linoleic acid and the common dietary antioxidant quercetin (flavonoid), both bound to human serum albumin (HSA). In particular, it is shown that quercetin, although embedded in drug site I, is oxidized as quickly as free quercetin under a flux of hydrophilic peroxyl radicals. This observation suggests that efficient charge relays are established between the periphery of HSA and bound quercetin. Moreover, the peroxidation of HSA-bound linoleic acid is shown to take place at some specific fatty acid binding sites once one to two critical HSA residues are themselves oxidized. Quercetin efficiently delays the onset of lipid peroxidation. The inhibition persists long after the total consumption of quercetin, in agreement with some quercetin oxidation products exerting a residual antioxidant activity. Consistently, HSA markedly increases the maximal concentration of a two-electron oxidation product of quercetin that is accumulated and then consumed in the course of the peroxidation. The additional observation of the faster consumption of the single Trp residue in the presence of quercetin suggests that HSA enhances the antioxidant activity of quercetin by regenerating some of its oxidation products retaining a H-donating activity.     

Understanding the effects of two bound glucose in Sudlow site I on structure and function of human serum albumin: theoretical studies

Human serum albumin (HSA) is the most abundant protein found in blood serum. It carries essential metabolites and many drugs. The glycation of HSA causes abnormal biological effects. Importantly, glycated HSA (GHSA) is of interest as a biomarker for diabetes. Recently, the first HSA structure with bound pyranose (GLC) and open-chain (GLO) glucose at Sudlow site I has been crystallised. We therefore employed molecular dynamics (MD) simulations and ONIOM calculations to study the dynamic nature of two bound glucose in a pre-glycated HSA (pGHSA) and observe how those sugars alter a protein structure comparing to wild type (Apo) and fatty acid-bound HSA (FA). Our analyses show that the overall structural stability of pGHSA is similar to Apo and FA, except Sudlow site II. Having glucose induces large protein flexibility at Sudlow site II. Besides, the presence of glucose causes W214 to reorient resulting in a change in W214 microenvironment. Considering sugars, both sugars are exposed to water, but GLO is more solvent-accessible. ONIOM results show that glucose binding is favoured for HSA (?115.04 kcal/mol) and GLO (?85.10 kcal/mol) is more preferable for Sudlow site I over GLC (?29.94 kcal/mol). GLO can strongly react with K195 and K199, whereas K195 and K199 provide slightly repulsive forces for GLC. This can confirm that an open-chain GLO is more favourable inside a pocket.     

Effect of lysine modification on the conformation and indomethacin binding properties of human serum albumin.

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in alpha-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.