Allosteric modulation of anti-HIV drug and ferric heme binding to human serum albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme-HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme-HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Resveratrol binding to human serum albumin

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Stereoselective binding of human serum albumin

Stereoselectivity in binding can have a significant effect on the drug disposition such as first-pass metabolism, metabolic clearance, renal clearance, and protein and tissue binding. Human serum albumin (HSA) is able to stereoselectively bind a great number of various endogenous and exogenous compounds. Various experimental data suggested that the two major drug-binding cavities, namely, site I and site II, do not seem to be the stereoselective binding sites of HSA. Stereoselective binding of HSA under disease conditions such as renal and hepatic diseases was found to be enhanced. In addition, site-to-site displacement of a site II-specific drug by another site II-specific drug was found to be stereoselective, too. Endogenous compounds such as long-chain fatty acids and uremic toxins are likely to cause combined direct and cascade effects that contribute to the preferential binding of a particular drug enantiomer. Taking together the findings of other studies, it is highly possible that the stereoselective binding site exists at the interface of the subdomains.     

Spectrophotometric and kinetic studies on the binding of the bioflavonoid quercetin to bovine serum albumin

This work investigates the binding of the bioflavonoid, quercetin, to bovine serum albumin (BSA) by spectrophotometric techniques involving both the conventional and stopped-flow methods. Both the neutral and negatively-charged forms of quercetin bound to BSA with a red shift in the maximal absorption. At high pH values, quercetin was rapidly degraded in an oxygen-dependent process, but this decomposition was substantially slower when the flavonoid was bound to BSA. At pH 7.4, the difference spectrum of quercetin with and without BSA was maximal at 425 nm; this wavelength can be conveniently used to monitor the extent and speed of binding. Spectrophotometric studies with a range of equimolar mixtures of quercetin and BSA at pH 7.4 suggest the binding was maximal when the concentration was 10 microM. It is postulated that the binding site of BSA for quercetin was less available at higher protein concentrations, perhaps because of conformational change or self-association. The rate of spectrophotometric change when quercetin bound to BSA was fairly slow; the process was not quite complete within 45 seconds and was biphasic. When a pre-mixed equimolar mixture of BSA and quercetin was diluted with an equal volume of the buffer, there was a surprising further increase in absorbance at 425 nm (rather than the fall anticipated if the binary complex were to dissociate). It is concluded that, upon dilution, the effective concentration of BSA's binding site increased, providing more scope for quercetin to bind.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Stereoselective binding of carbenicillin epimers to human serum albumin

Binding of carbenicillin (CBPC) epimers to human serum albumin (HSA) was found to be stereoselective. Epimer-epimer interaction was also observed in the binding to HSA. There were at least three binding sites on HSA for CBPC epimers, one of which (stereoselective site) was more in favor of S-CBPC than R-CBPC. At the stereoselective site, the binding constant of S-CBPC was approximately 4-fold greater than that of R-CBPC. The affinities to other binding sites (non-stereoselective sites) were similar between the epimers, and the affinity of S-CBPC of the non-stereoselective sites was much smaller than that for the stereoselective site. R-CBPC and S-CBPC appeared to displace each other at all the binding sites, i.e., the binding of the epimers was competitive at the non-stereoselective sites as well as at the stereoselective site. By using site marker ligands, it was revealed that CBPC epimers may bind to Site I (warfarin binding site), but not to Site II (diazepam binding site). A binding model with an assumption of competitive interactions at all the binding sites simulated the binding characteristics of CBPC epimers fairly well. © 1996 Wiley-Liss, Inc.     

Chromatographic analysis of carbamazepine binding to human serum albumin

In this study, high-performance affinity chromatography was used to characterize the binding of carbamazepine to an immobilized human serum albumin (HSA) column. Frontal analysis was first used to determine the association equilibrium constant and binding capacity for carbamazepine on this column at various temperatures. The non-specific binding of carbamazepine within the column was also considered. The results indicated that carbamazepine had a single binding site on HSA with an association equilibrium constant of 5.3 x 10(3)M(-1) at pH 7.4 and 37 degrees C. This was confirmed through zonal elution self-competition studies. The value of DeltaG for this reaction was -5.35 kcal/mol at 37 degrees C, with an associated change in enthalpy (DeltaH) of -6.45 kcal/mol and a change in entropy (DeltaS) of -3.56 cal/molK. The location of this binding region was examined by competitive zonal elution experiments using probe compounds with known sites on HSA. It was found that carbamazepine had direct competition with l-tryptophan, a probe for the indole-benzodiazepine site of HSA, but allosteric interactions with probes for the warfarin, tamoxifen and digitoxin sites. Changes in the pH, ionic strength, and organic modifier content of the mobile phase were used to identify the predominant forces in the carbamazepine-HSA interaction.     

Study of heterocycle rings binding to human serum albumin

Doxorubicin continues to be one of the most widely used anticancer agents in the clinic despite its dose-limiting side-effects. Many of doxorubicin's dose-limiting toxicities occur due to its generation of toxic oxygen species, resulting in oxidative stress. Some clinical observations have suggested that doxorubicin may have greater toxicity in older patients. The studies presented here compare basal and doxorubicin-induced antioxidant enzyme activities in brain, heart, kidney and liver tissues of Fisher 344 rats of different ages to determine whether differences in these enzymes can account for the age-dependent differences observed in doxorubicin-induced toxicity. Three groups of animals were tested, young animals (2-months-old), adult animals (10-months-old) and old animals (18-months-old). The results of these studies show that in general young and adult animals have similar levels of antioxidant enzyme activity while the older animals have less. Only in the young animals is antioxidant enzyme activity significantly increased following doxorubicin treatment suggesting that enzyme induction occurs only in the young group of animals. Lipid peroxidation is shown to have the greatest increase in the old animals following doxorubicin treatment while the young animals have the smallest increase. The results from these studies suggest that there is an increase in doxorubicin-induced oxidative damage with age and that these differences may be due to basal and drug-induced differences in tissue antioxidant enzyme activities.     

Binding of quercetin with human serum albumin: a critical spectroscopic study

Flavonols are plant pigments that are ubiquitous in nature. Quercetin (3,3',4',5,7-pentahydroxyflavone) and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, antitumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. Quercetin is intrinsically weakly fluorescent in aqueous solution, showing an emission maximum at approximately 538 nm. Upon binding to human serum albumin (HSA), quercetin undergoes dramatic enhancement in its fluorescence emission intensity, along with the appearance of dual emission behavior, consisting of normal and excited-state proton transfer (ESPT) fluorescence. In addition, the occurrence of a third emitting species has been noted for the first time. This is attributed to a electronic ground-state complex formed in the protein environment. High values of the fluorescence anisotropy (r) are obtained in the presence of HSA for the ESPT tautomer (r = 0.18), as well as the complex species (r = 0.37) of quercetin, indicating that the precursor ground-state molecules for both these emitting species of quercetin molecules are located in the motionally constrained sites of HSA. The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place. The preliminary results of studies on emission decay kinetics are also reported herein. Studies by far-UV circular dichroism spectroscopy reveal that binding of quercetin induces no significant perturbation in the secondary structure of HSA.     

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.     

Data plotting of warfarin binding to human serum albumin

The binding of warfarin to human serum albumin was studied by equilibrium dialysis at pH 7.4 in a 67 mM sodium phosphate buffer at 37 degrees C. The equilibrium data were analysed using a computer program for curve fitting. The analysis was made fitting the data to equations for one, two and three classes of binding sites with one, two and three sites at the primary binding site (n(1)=1, 2 or 3). The data fitting was acceptable for two and three classes of binding sites but the best fit was obtained with the equation for two classes of binding sites, allowing us to define the binding by a model with two independent classes of binding sites on the serum albumin molecule.     

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.     

Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

The specific binding of chlorogenic acid to human serum albumin

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in human diet. It is also an active component in traditional Chinese medicines which are used to treat various diseases. In this study, fluorescence spectroscopy in combination with UV–Vis absorption spectroscopy was employed to investigate the specific binding of CGA to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by CGA is a result of the formation of CGA–HSA complex. Binding parameters calculating from Stern–Volmer method and Scatchard method showed that CGA bind to HSA with the binding affinities of the order 10⁴ l mol⁻¹. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for CGA–HSA association. Site marker competitive displacement experiments demonstrated that CGA specific bind to site I (subdomain IIA) of HSA. The binding distance r (3.10 nm) between donor (Trp-214) and acceptor (CGA) was obtained according to fluorescence resonance energy transfer. Furthermore, the effect of metal ions on CGA–HSA system was studied.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.     

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR-albumin complexes. Photoirradiation results of BR-albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.     

High affinity binding of paclitaxel to human serum albumin.

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle-related toxicity, we developed a novel, water-soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel-albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP-HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 x 10(6) M(-1)). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 x 10(6) M(-1) and K2 = 1.0 x 10(5) M(-1)). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.