Temperature dependence on the binding of butyl orange by bovine serum albumin: The effect of urea

The effect of urea on the extent of the binding of butyl orange by bovine serum albumin has been examined by an equilibrium dialysis method. The first binding constants and the thermodynamic parameters for the formation of the first dye anion–protein complex have been calculated. Addition of urea to the binding system causes a marked decrease in the absolute magnitude of the free energy change. The enthalpy change during binding becomes more exothermic, and the entropy change tends to decrease with increasing concentration of urea. These results can be interpreted in terms of the concept that urea reduces the structure of the aqueous environment and hence lowers the tendency of apolar groups of the dye and the albumin to participate in the formation of hydrophobic interactions.     

Investigation of the interaction between acridine orange and bovine serum albumin

The results from the measurement of the fluorescence spectrum showing the binding characteristics of acridine orange (AO) and bovine serum albumin (BSA) are reported. It was found that the equilibrium constant k=4848.64 l mol⁻¹, and the number of binding sites n=0.82. Based on the mechanism of the Forster energy transference, the transfer efficiency of energy and distance between the acceptor AO and BSA were found. The interaction between AO and BSA have been verified as consistent with the static quenching procedure and the quenching mechanism is related to the energy transfer.     

Study on the binding of luteolin to bovine serum albumin

Binding of luteolin (LU) to bovine serum albumin (BSA) was investigated at 298, 308 and 318K at pH 7.4 using spectrophotometric techniques such as fluorescence emission, circular dichroism (CD). The data obtained from fluorescence quenching experiments showed that LU was bound to BSA and binding constants and the number of binding sites (n approximately 1) were obtained. The thermodynamic parameters DeltaH(0), DeltaS(0), DeltaG(0) at different temperatures were calculated. They indicated that both hydrophobic forces and hydrogen bonds are the major interactions between LU and BSA. A value of 3.12nm for the average distance r between LU (acceptor) and tryptophan residue (Trp) of BSA (donor) was derived from the fluorescence resonance energy transfer. The effects of some common metal ions on the binding are also considered. Besides, the interaction of BSA with LU led to a change in the conformation of BSA.     

Gadolinium and calcium binding to bovine serum albumin

The temperature jump relaxation technique is used to study the binding of calcium(II) and gadolinium(III) to bovine serum albumin at 7°C and an ionic strength of 0·2 (NaClO₄). Both metal ions are predicted to bind to the ϵ-amino groups of the protein above pH = 8·2, where extensive metal ion hydrolysis takes place. For the reaction between GdOH²⁺ and bovine serum albumin, the complex formation rate constant is (1·0±0·1) × 10⁸ M⁻¹ sec⁻¹ and the dissociation rate constant is (9·3±2·1) sec⁻¹. The corresponding calcium association reaction is too rapid to obtain rate constants. The complex formation rate constant is compared to other gadolinium systems to explain the relatively high complexation constant with bovine serum albumin.     

Thyroxine binding properties of glycosylated bovine serum albumin.

Thyroid hormone, thyroxine (T4) binding properties of glycosylated bovine serum albumin (G-BSA), and intact BSA were studied by the fluorescence method. The apparent binding constants for intact BSA were 0.8 (0.16) x 10(6) M-1 at pH 5.0 and 2.18 (0.06) x 10(6) M-1 at pH 9.5 at 25 degrees C. T4 binding for G-BSA was independent of pH and the apparent binding constant was 1.4 x 10(6) M-1. Thermodynamic parameters were also evaluated from the Van't Hoff plots of the apparent binding constants at pH 7.4 and 8.5. At both pH's, the free energy, enthalpy and entropy changes were almost the same for both G-BSA and BSA.     

Thermodynamics of binding cationic detergents to bovine serum albumin

Molar ratio (Γ_a) for binding cetyl trimethyl ammonium bromide (CTAB), myristyl trimethyl ammonium bromide (MTAB), dodecyl trimethyl ammonium bromide (DTAB) and cetyl pyridinium chloride (CPCL) to globular protein bovine serum albumin have been measured by equilibrium dialysis technique under various conditions of pH, ionic strength and temperatures. In all cases binding isotherms are S-shaped and at high concentrations of an amine, Γ_a appears to reach a constant value Γ^m_a, Γ^m_a may vary from 1 to 836 depending upon temperature, nature of amines and other solution parameters. The standard free energy changes (ΔG°) for BSA-amine binding have been calculated using appropriate reference states. The order of ΔG° for various systems is found to be in agreement with the order of the maximum extent of binding Γ^m_a. The effects of chain length of the surfactants on ΔG° and Γ^m_a are found to be irregular. ΔS° and ΔH° for binding reactions are also found to be significant in magnitude. Binding interactions at various pH and ionic strengths do not exhibit regularities in terms of electrostatic effects. All these observations lead to the conclusion that the binding sites of BSA are highly heterogeneous so that binding interactions become irregular in nature. All types of interactions are involved in the binding process so that the mechanism of binding in an individual system is complex in nature. The thermal stability and intrinsic viscosity of bovine serum albumin in the presence of amines have been examined in the light of the binding process.     

荧光光谱法研究左西孟旦与牛血清白蛋白的结合反应

用荧光光谱法、分光光度法研究了水溶液中左西孟旦与牛血清白蛋白(BSA)的相互结合反应。研究表明:左西孟旦对BSA的内源荧光有较强的猝灭作用且该猝灭作用属于静态荧光猝灭作用。得出了反应的结合常数(KA=1.48×106L/mol)和结合位点数(n=1.14)。根据Frster非辐射能量转移机理,求算了给体(BSA)与受体(左西孟旦)间距离r=2.9 nm和能量转移效率E=0.33。     

Study on the binding behavior of bovine serum albumin with cephalosporin analogues by chemiluminescence method

The luminol-bovine serum albumin chemiluminescence system was proposed for the first time. It was found that the hydrophilic luminol bound to the hydrophilic domain at Trp¹³⁴ of BSA with accelerating the electrons transferring rate of excited 3-aminophthalate, which led to the enhancement CL intensity of luminol at 425 nm. The increment of chemiluminescence intensity was proportional to the concentrations of bovine serum albumin from 5.0 × 10⁻¹¹ to 1.0 × 10⁻⁸ mol L⁻¹ with the linear equation of ΔI = 7.47C_BSA + 4.89 (R² = 0.9950). Based on the remarkable quenching effect of cephalosporin on the luminol-bovine serum albumin chemiluminescence system, the interaction of bovine serum albumin-cephalosporin was studied by flow injection-chemiluminescence method. A valuable model for studying the interaction of bovine serum albumin-cephalosporin was constructed and the formula lg[(I₀ − I)/I] = lg K_D + n lg[D] was obtained. The binding parameters calculated by the model did agree very well with the results obtained by fluorescence quenching method. The major binding force of bovine serum albumin with cephalosporins was the hydrophobic effect. The binding ability of cephalosporin analogues to bovine serum albumin followed the pattern: cefoperazone, ceftriaxone and cefotaxime > cefuroxime and cefaclor > cefadroxil, cefradine and cefazolin, which was close to the order of their antibacterial ability. Using flow injection chemiluminescence method also obtained the stoichiometric ratio, the average of association constant K_P and dissociation degree α of luminol-bovine serum albumin were 1:1, 1.12 × 10⁷ L mol⁻¹ and 0.086, respectively.     

Temperature dependence of solvation dynamics and anisotropy decay in a protein: ANS in bovine serum albumin

Temperature dependence of solvation dynamics and fluorescence anisotropy decay of 8-anilino-1-naphthalenesulfonate (ANS) bound to a protein, bovine serum albumin (BSA), are studied. Solvation dynamics of ANS bound to BSA displays a component (300 ps) which is independent of temperature in the range of 278-318 K and a long component which decreases from 5800 ps at 278 K to 3600 ps at 318 K. The temperature independent part is ascribed to a dynamic exchange of bound to free water with a low barrier. The temperature variation of the long component of solvation dynamics corresponds to an activation energy of 2.1 kcal mol(-1). The activation energy is ascribed to local segmental motion of the protein along with the associated water molecules and polar residues. The time scale of solvation dynamics is found to be very different from the time scale of anisotropy decay. The anisotropy decays are analyzed in terms of the wobbling motion of the probe (ANS) and the overall tumbling of the protein.     

Heterotropic binding of alclofenac and dansylsarcosine to bovine serum albumin

The binding data for the interaction of alclofenac (AF) and dansylsarcosine (DS) to bovine serum albumin (BSA) have respectively yielded nonlinear Scatchard plots. The plots have been subjected to Rosenthal’s method of analysis and thus the ligands have been found to possess two different kinds of sites in BSA. The binding capacities of these sites have been evaluated. The fluorescence competition studies have revealed that the binding of DS to BSA is noncompetitively inhibited by AF. Therefore, the presence of distinct binding sites for AF and DS in BSA could be inferred. The fluorescence quenching studies have also been able to demonstrate this aforesaid fact. The analysis of the quenching data by the modified Stern-Volmer plot has indicated that both the tryptophan (Trp) residues of BSA are accessible to DS for the quenching in absence of AF, but only one of them is accessible in presence of AF. This has led to suggest that the binding site of DS has been in the vicinity of loop 3–4, involving Trp-134 and Trp-212. The binding of AF at a distinct site from that of DS has exerted heterotropic interactions at the DS binding site and thereby inhibited the binding of DS to BSA.     

Characterization of hydroxycinnamic acid derivatives binding to bovine serum albumin

Hydroxycinnamic acid derivatives (HCAs) are a group of naturally occurring polyphenolic compounds which possess various pharmacological activities. In this work, the interactions of bovine serum albumin (BSA) with six HCA derivatives, including chlorogenic acid (CHA), caffeic acid (CFA), m-coumaric acid (m-CA), p-coumaric acid (p-CA), ferulic acid (FA) and sinapic acid (SA) have been investigated by NMR spectroscopic techniques in combination with fluorescence and molecular modeling methods. Competitive STD NMR experiments using warfarin sodium and L-tryptophan as site-selective probes indicated that HCAs bind to site I in the subdomain IIA of BSA. From the analysis of the STD NMR-derived binding epitopes and molecular docking models, it was deduced that CHA, CFA, m-CA and p-CA show similar binding modes and orientation, in which the phenyl ring is in close contact with protein surface, whereas carboxyl group points out of the protein. However, FA and SA showed slightly different binding modes, due to the steric hindrance of methoxy-substituents on the phenyl ring. Relaxation experiments provided detailed information about the relationship between the affinity and structure of HCAs. The binding affinity was the strongest for CHA and ranked in the order CHA > CFA > m-CA ≥ p-CA > FA > SA, which agreed well with the results from fluorescence experiments. Based on our experimental results, we also conclude that HCAs bind to BSA mainly by hydrophobic interaction and hydrogen bonding. This study therefore provides valuable information for elucidating the mechanisms of BSA-HCAs interaction.     

Binding of naproxen to bovine serum albumin and tryptophan-modified bovine serum albumin

Two classes of binding sites, a single high-affinity site with an association constant of 4·8×10⁶ M⁻¹ and two low-affinity sites with association constant of about 0·05×10⁶ M⁻¹ have been observed in the interaction of Naproxen with bovine serum albumin (BSA). Chemical modification of two tryptophan residues in BSA with 2-hydroxy-5-nitrobenzyl bromide has led to a reduction in the association constant of the high-affinity site by 89% and its number of binding sites by 66% suggesting the involvement of tryptophan residues in the high-affinity site. In contrast, the two low-affinity sites were not affected by the modification. Binding of Naproxen to the low-affinity sites of BSA induces microdisorganisation of the albumin structure leading to conformational changes as evident from fluorescence measurements with 1-anilino-8-naphthalenesulphonic acid as the probe.     

Viscoelastic study of the adsorption of bovine serum albumin on gold and its dependence on pH

Using a quartz crystal resonator system operating at 5 MHz the shear wave propagating properties of bovine serum albumin (BSA) have been monitored as it is adsorbed on a gold surface from a phosphate buffered saline (PBS) solution. Employing a 2-layer model for the combined BSA layer and PBS solution, the viscoelasticity of the BSA layer may be determined in real time as the adsorption on gold proceeds. The viscoelasticity is found to depend on the pH of the PBS solution and changes gradually over long times. It is suggested that at the low frequency of the measurement, large-scale molecular motions are being monitored which are a consequence of the structural changes in the protein molecules undergoing adsorption. Such low-frequency molecular motions are difficult to examine by any other technique. The results and their interpretation in viscoelastic terms demonstrate the considerable potential of the quartz crystal resonator system for assessing the stability of proteins on surfaces and their suitability as coatings for prosthetic materials.     

Thermal denaturation of bovine serum albumin and its oligomers and derivativespH dependence

In a previous paper, we report a preliminary DSC study on bovine (BSA) and human (HSA) serum albumins. However, at accurate HPLC analysis the commercial proteins show three peaks: Fraction V-I, probably globulins (as declared by the producers), Fraction V-II (about 15–18% of the product) and Fraction V-III that represents pure BSA or HSA. A hypothesis is that the Fraction II is a covalent dimer, or trimer or a mixture of both, generated during the scalf-life of the commercial product. Denaturation enthalpies of the purified Fraction V-III and Fraction V-II of BSA, have been determined calorimetrically, at changing thepH, and the results of both compared with those obtained on the untreated protein. Few calorimetric experiments have been also carried on a BSA monomer derivative with sulphidril group protected. Computer program have been developed for the deconvolution of exo- and endothermic effects and for the analysis of thermal denaturation profiles.     

Study on the binding of puerarin to bovine serum albumin by isothermal titration calorimetry and spectroscopic approaches

The interaction of a flavonoid molecule (puerarin) with bovine serum albumin (BSA) was characterized by isothermal titration calorimetry (ITC), optical spectroscopic technique, and molecular modeling method under physiological conditions. The binding parameters for the reaction were calculated according to ITC experiments at different temperatures. The thermodynamic parameters, negative enthalpy changes (ΔH), and positive entropy (ΔS) indicated that the binding processes were entropically driven. The alterations of protein secondary structure in the presence of puerarin in aqueous solution were estimated by the evidences from FT-IR and CD spectroscopy with reductions of α-helices. On the basis of fluorescence resonance energy transfer (FRET) between excited tryptophan in BSA and BSA bound puerarin, the critical transfer distance and mean distance between tryptophan in BSA and puerarin were estimated.     

Study on the structural changes of bovine serum albumin with effects on polydatin binding by a multitechnique approach

Polydatin is a traditional Chinese medicine which shows effective biological activity as antimicrobial and antiviral agent. The secondary structure changes of bovine serum albumin (BSA) were investigated by the methods of Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) and Raman spectroscopy. The experimental results indicated that polydatin changed the secondary structure of BSA. The presence of polydatin decreased α-helix content of BSA. The conformations of disulfide bridges and the microenvironment of Tyr, Trp residues were also changed.     

Mechanism and conformational studies of farrerol binding to bovine serum albumin by spectroscopic methods

The mechanism and conformational changes of farrerol binding to bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence quenching technique, UV–vis absorption, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that farrerol could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The thermodynamic parameters enthalpy change and entropy change for the binding were calculated to be −29.92 kJ mol⁻¹ and 5.06 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which suggested that the both hydrophobic interactions and hydrogen bonds play major role in the binding of farrerol to BSA. The binding distance r deduced from the efficiency of energy transfer was 3.11 nm for farrerol–BSA system. The displacement experiments of site markers and the results of fluorescence anisotropy showed that warfarin and farrerol shared a common binding site I corresponding to the subdomain IIA of BSA. Furthermore, the studies of synchronous fluorescence, CD and FT-IR spectroscopy showed that the binding of farrerol to BSA induced conformational changes in BSA.     

Spectroscopic study on the inherent binding information of cationic perfluorinated surfactant with bovine serum albumin

UV-vis, FT-IR, fluorescence and synchronous fluorescence spectra are applied to discuss the inherent binding information of model protein bovine serum albumin (BSA) with perfluorinated surfactant trimethyl-1-propanaminium iodide (FC-134). According to the results analyzed from Stern-Volmer equation, FC-134 can quench the fluorescence intensity of BSA via a dynamic quenching mechanism with complex formation. The thermodynamic parameters are calculated, revealing that hydrophobic force is the main interaction driven force. The binding constants and number of binding sites are also obtained. With the aid of site markers—warfarin and ibuprofen, we first report that FC-134 primarily binds to tryptophan residue Trp-214 of BSA within site I (sub-domain IIA).