Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements.

The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins.     

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.     

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.     

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.     

Experimental and computational studies on the binding of diazinon to human serum albumin

In the present research, the binding properties of diazinon (DZN), as an organophosphorus herbicide, to human serum albumin (HSA) were investigated using combination of spectroscopic, electrochemistry, and molecular modeling techniques. Changes in the UV–Vis and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. The obtained results from spectroscopic and electrochemistry experiments along with the computational studies suggest that DZN binds to residues located in subdomains IIA of HSA with binding constant about 1410.9 M^?1 at 300 K. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH° and entropy change ΔS° were found to be ?16.695 and 0.116 KJ/mol K, respectively. The primary binding pattern is determined by hydrophobic interaction and hydrogen binding occurring in so-called site I of HSA. DZN could slightly alter the secondary structure of HSA. All of experimental results are supported by computational techniques such as docking and molecular dynamics simulation using a HSA crystal model.     

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

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

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

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.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.     

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.     

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.     

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.     

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.