Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Binding of long-chain fatty acids to bovine serum albumin

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.     

Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis.

A quick and reproducible capillary electrophoresis method was optimized and validated for the assay of bovine serum albumin (BSA). The effects of various parameters such as pH of buffer, concentration of buffer, capillary dimensions, use of coated capillaries, and additives such as surfactants and protein solubilizers were evaluated. The capillary coatings or additives did not give any advantage in reducing the surface adsorption of BSA on the capillary walls. The optimized conditions include use of borate buffer, pH 8.5 having a concentration of 150 mM in a 27 cm capillary with an aperture window of 100 x 200 microns for detection. The optimized method for the detection of BSA was validated. The interday and intraday coefficient of variation was not greater than 7.59% at BSA concentrations of 25-1000 micrograms/ml. The method developed was reproducible and accurate.     

Binding of the environmental pollutant naphthol to bovine serum albumin

The interactions between naphthol and bovine serum albumin (BSA) were investigated by spectroscopy. Our results prove the formation of complex between naphthol and BSA. Hydrophobic interaction dominates in the association reaction. The isomers stack with the aromatic residues in their binding sites with different geometries. Effects of BSA on the excited-state proton transfer and fluorescence spectra of the isomers indicate the different characters of their binding sites. 1-Naphthol inserts deeply into a hydrophobic cavity whereas 2-naphthol is in a basic environment on the surface of BSA. Naphthol statically quenches the fluorescence of BSA in a concentration-dependent manner positively deviating from the linear Stern-Volmer equation. Naphthol binds near Trp-134 in the subdomain IA of the native BSA and is accessible to Trp-212 when BSA is unfolded by naphthol. The folding pattern of the main chain is altered at high naphthol concentration as revealed by the change in the secondary structure. The binding of 1-naphthol is more cooperative than that of 2-naphthol. The extent of cooperativity was estimated by the Hill equation.     

Binding of anti-inflammatory drug cromolyn sodium to bovine serum albumin

Fluorescence spectroscopy in combination with circular dichroism (CD) and UV–vis absorption spectroscopy were employed to investigate the binding of anti-inflammatory drug cromolyn sodium (Intal) to bovine serum albumin (BSA) under the physiological conditions with Intal concentrations of 0–6.4 × 10⁻⁵ mol L⁻¹. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by Intal is a result of the formation of Intal–BSA complex. Quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between Intal and BSA. The thermodynamic parameters ΔG, ΔH, ΔS at different temperatures (298, 304, and 310 K) were calculated and the results indicate the electrostatic interactions play a major role in Intal–BSA association. Binding studies concerning the number of binding sites (n = 1) and apparent binding constant K_b were performed by fluorescence quenching method. Utilizing fluorescence resonant energy transfer (FRET) the distance R between the donor (BSA) and acceptor (Intal) has been obtained. Furthermore, CD and synchronous fluorescence spectrum were used to investigate the structural change of BSA molecules with addition of Intal, the results indicate that the secondary structure of BSA molecules was changed in the presence of Intal.     

Binding of berberine to bovine serum albumin: spectroscopic approach

Fluorescence spectroscopy in combination with UV–vis absorption spectroscopy was employed to investigate the binding of an important traditional medicinal herb berberine to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by berberine is a result of the formation of berberine–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS at different temperatures indicate that the electrostatic interactions play a major role for berberine–BSA association. Site marker competitive experiments indicated that the binding of berberine to BSA primarily took place in site II. Furthermore, the Effect of supramolecules to berberine–BSA system, and the distance r between donor (BSA) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).     

Binding of lead ion to bovine serum albumin studied by ion selective electrode

The binding of Pb2+ to bovine serum albumin (BSA) at neutral pH was studied using lead ion selective electrode. The binding data was treated according to Scatchard Equation. The number of binding classes and the number of binding sites, intrinsic dissociation constants and stepwise binding constants for each class were determined. Two binding classes were found. Four binding sites in the first class and five binding sites in the second class were determined. Binding in the first class was stronger than in the second. Similar binding studies were carried out with heat treated BSA. It was found that not only the number of binding sites but also the strength of binding increases upon heat treatment.     

Binding of bromophenol blue to bovine serum albumin and its succinylated forms

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformational changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused significant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Interaction of zearalenone with bovine serum albumin as determined by fluorescence quenching

The major aim of this study was to examine the binding of zearalenone (ZEN) to bovine serum albumin (BSA) by measuring the quenching of the intrinsic fluorescence of the protein under aqueous conditions. The results suggest that ZEN has a strong ability to quench the intrinsic fluorescence of BSA through a static mechanism. The hydrophobicity of the microenvironment around the tyrosine (Tyr) residues in BSA was increased in the presence of ZEN. The quenching constants, ratio of protein with ZEN, and thermodynamic parameters were determined. The collaborative action of hydrophobic and electrostatic interactions was involved in the binding process and the formation of the complex was mainly enthalpy-driven. The average binding distance between ZEN and BSA was calculated to be 2.20 nm. This is much closer in magnitude than the distance reported for the binding of most toxins to HSA and most pharmaceuticals to BSA, indicating a strong affinity.     

Binding of Triton X-100 to bovine serum albumin as studied by surface tension measurements

A previously published computerized drop-weight technique for surface tension measurements, not involving the use of radioactively labelled compounds, has been applied to the study of detergent binding to proteins. The procedure is based on the observation that the protein-surfactant complex is no longer surface-active. As an example, the binding of Triton X-100 to bovine serum albumin has been studied, and the results were found to be in good agreement with those obtained through established but less convenient methods. Our procedure should be useful for measurements of detergent binding to biomembranes.     

Characterization of polyanion-protein complexes by frontal analysis continuous capillary electrophoresis and small angle neutron scattering: effect of polyanion flexibility

The binding constant (K(obs)) for the beta-lactoglobulin-poly(vinylsulfate) (BLG-PVS) complex was measured by frontal analysis continuous capillary electrophoresis at pH values above the isoelectric point of BLG, and the persistence length (L(p)) of PVS was measured by small angle neutron scattering, to examine the effect of polyelectrolyte chain stiffness on its binding efficiency to proteins. The values of K(obs) and L(p) were compared with those of BLG-PSS and BLG-PAMPS (poly(2-acrylamido-2-methylpropanesulfonate)) reported previously. The relationship between K(obs) and L(p) was reciprocal, indicating that protein binding is enhanced by the flexibility of the polyanion, at least in the case where the net protein charge is negative. In addition, at a fixed pH, the polymer systems displayed a similar ionic strength dependence of K(obs). This similarity was consistent with the proposal that the binding properties of PVS and PAMPS polyanions are governed purely by electrostatic interactions and are independent of their molecular structure.     

DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.