Application of affinity capillary electrophoresis for the determination of binding and thermodynamic constants of enediynes with bovine serum albumin

The binding constants and thermodynamic properties of a series of novel enediyne compounds with bovine serum albumin (BSA) were determined. The enediynes were synthesized, characterized, and then studied by affinity capillary electrophoresis (ACE) methods to derive these recognition parameters. Change in electrophoretic mobility of BSA as a function of enediyne concentration was determined at 25 degrees C providing binding constants of 1.76 x 10(5), 1.14 x 10(5), and 0.68 x 10(5) M(-1) for enediynephenylalanine carboxylic acid, enediynephenylalanine methyl ester, and enediyne carboxylic acid, respectively. The binding constant for the enediynephenylalanine carboxylic acid was in good agreement with that obtained using conventional methodology. Binding constants for the interaction of enediynes with BSA decreased with an increase in temperature. Van't Hoff plots showed a direct correlation between intensity of the binding constant and the sign and magnitude of various thermodynamic parameters (DeltaG, DeltaS, and/or DeltaH).     

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.     

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).     

毛细管区带电泳法测定4种抗HIV-1活性的化合物与牛血清白蛋白的结合常数

采用毛细管区带电泳紫外检测方法,在pH 8.0、浓度50 mmol/L的磷酸盐缓冲溶液及运行电压15 kV的条件下,测定了4种新合成的具有抗HIV-1活性的化合物(IG3,iso-C3,C3,MC3)与牛血清白蛋白(BSA)相互作用的结合常数。在缓冲溶液中加入不同浓度的BSA,通过测定化合物迁移时间的变化,计算得到了上述4种化合物与BSA的结合常数分别为1.07×104, 1.34×104, 8.51×103和9.45×103 L/mol。该方法简单、快捷,可用于研究结合比为1∶1的小分子与生物大分子的     

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.     

Characterization of the interaction between a new merocyanine dye and bovine serum albumin

A new merocyanine dye was synthesized, and its acidity constant was determined by spectrophotometric and chemometrics methods. The interactions of the new cyanine dye with bovine serum albumin (BSA) have been studied by fluorescence and UV absorption spectroscopy at pH 7.40. A visual color change from red to blue was observed by addition of BSA to aqueous solution of the dye. The quenching constants and binding parameters (binding constants and number of binding sites) were determined at different temperatures. The calculated thermodynamic parameters confirmed that the binding reaction is mainly entropy-driven, whereas electrostatic interaction plays major role in the reaction. The displacement experiment confirmed binding of the dye to the subdomain IIA (site 1) of albumin. Moreover, synchronous fluorescence spectroscopy studies revealed the dye induces some local conformational change in BSA. The binding distance, r, between donor (serum albumin) and acceptor (dye) was obtained according to Förster’s theory.     

Binding of the bioactive component daphnetin to human serum albumin demonstrated using tryptophan fluorescence quenching

Daphnetin (7,8-dihydroxycoumarin), one of the major bioactive components isolated from Daphne koreane Nakai, has been used in traditional Chinese medicine for the treatment of coagulation disorders. It is also a chelator, an antioxidant and a protein kinase inhibitor. In this paper, a combination of intrinsic fluorescence, Fourier transform infrared (FT-IR) spectroscopy and circular dichroic (CD) spectroscopy has been used to characterize the binding between daphnetin and human serum albumin (HSA) under physiological conditions with drug concentrations of 6.7 x 10(-6) - 2.3 x 10(-5) mol x L(-1), and a HSA concentration of 1.5 x 10(-6) mol x L(-1). Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching did change significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated to be -12.45 kJ x mol(-1)and 52.48 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic and electrostatic interactions played the main role in the binding of daphnetin to HSA, in accordance with the results of calculations performed on a Silicon Graphics Ocatane2 workstation. In addition, the binding distance between daphnetin and HSA was obtained (4.02 nm) based on the Forster energy transfer theory.     

A kinetic study of protein binding to ecabet sodium using quartz-crystal microbalance.

To define the mechanism of the protection by ecabet sodium of the gastric mucosa, the characteristics of protein binding of this drug were investigated using a quartz-crystal microbalance (QCM) method. The binding rate constants (kb) and the binding amounts (delta m) were obtained from time courses of the frequency decrease (mass increase) of the QCM. The binding constants to proteins of two ecabet analogues (G1, ecabet type and G2, non-ionic ecabet type) were dependent on the pH, leading to large kb values at the acidic region. Furthermore, the kb values of G1 with the addition of bovine serum albumin (BSA) and bovine serum fibrinogen (BSF) at the acid region were larger than those of G2. The difference in kb values between G1 and G2 for porcine gastric mucin (PGM) is hardly discernible. Ecabet seems to be more heavily distributed in the ulcerous areas than in the intact mucosa, judging from the large binding constants of this drug to BSA and BSF compared with those to PGM. It is suggested that ecabet is bound to proteins by hydrophobic interaction, moreover, the electrostatic interaction between this drug and proteins (BSA and BSF) occurs at acidic pH region. On account of these interactions, ecabet sodium seems to have a more protective effect on an ulcer at intraluminal acidity than sucralfate. Finally, QCM was found to be a useful technique for detecting quantitatively the time course of binding proteins with drug.     

Study of the interaction of carbamazepine with bovine serum albumin by fluorescence quenching method.

The interaction between carbamazepine (CBZ) and bovine serum albumin (BSA) was studied using fluorescence spectroscopy (FS) and ultraviolet spectroscopy (UV). The experimental results showed that the CBZ could insert into the BSA and quench the inner fluorescence of BSA by forming the CBZ-BSA complex. It was found that both static quenching and non-radiation energy transfer were the main reasons leading to the fluorescence quenching. The apparent binding constants (K) between CBZ and BSA were found to be 1.8 x 10(4) (27 degrees C) and 2.8 x 10(4) (37 degrees C) and the binding site values (n) were 0.97 (27 degrees C) and 1.01 (37 degrees C), respectively. According to the Forster theory of non-radiation energy transfer, the binding distances (r) between CBZ and BSA were 3.6 nm and 3.4 nm at 27 degrees C and 37 degrees C, respectively. The process of the binding was a spontaneous molecular interaction in which entropy increased and Gibbs free energy decreased, indicating that the interaction between CBZ and BSA was mainly driven by the hydrophobic force.     

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.     

Spectroscopic studies on the mode of interaction of an anticancer drug with bovine serum albumin

The mechanism of interaction of vinblastin sulphate (VBS) with bovine serum albumin (BSA) has been reported. Association constant for VBS-BSA binding was found to be 3.146+/-0.06 x 10(4) M(-1). Stern-Volmer analysis of fluorescence quenching data showed that the fraction of fluorophore (protein) accessible to the quencher (drug) was close to unity indicating thereby that both tryptophan residues of BSA are involved in drug-protein interaction. The rate constant for quenching, greater than 10(10) M(-1) S(-1), indicated that the drug-binding site is in close proximity to tryptophan residues of BSA. Binding studies in the presence of an hydrophobic probe, 8-anilino-1-naphthalein-sulphonic acid, sodium salt (ANS) indicated that there is hydrophobic interaction between VBS and probe and they do not share common sites in BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of VBS to BSA involves predominant hydrophobic forces. The effects of some additives and paracetamol on binding of VBS-BSA have also been investigated. The CD spectrum of BSA in presence of VBS shows that the binding of VBS leads to change in the helicity of BSA.     

Study on the Interaction Between Bovine Serum Albumin and Potassium Perfluoro Octane Sulfonate

The interactions between potassium perfluorooctanesulfonate (PFOS) and bovine serum albumin (BSA) were studied by fluorescence spectroscopy. The association constants between PFOS and BSA were obtained by fluorescence enhancing and fluorescence quenching respectively. Furthermore, fluorescence quenching was studied at different temperatures, and the binding constant was also determined by the method of fluorescence quenching. According to the thermodynamic parameters, the main binding force could be judged. The experimental results revealed that BSA and PFOS had strong interactions. The mechanism of quenching belonged to dynamic quenching and the main sort of binding force was hydrophobic force. IR-spectra proved the interaction changed the conformation of BSA.     

Bonding strength of fluorinated and hydrogenated surfactant to bovine serum albumin

The interactions between bovine serum albumin (BSA) and different surfactants are investigated by the fluorescence technique. Pairs of fluorinated and hydrogenated surfactants with similar hydrophobic chain lengths including potassium perfluorooctanesulfonate and sodium octanesulfonate are studied in order to determine their interactions with BSA. The binding constants and thermodynamic parameters between BSA and different surfactants are compared and the main binding strength is determined. The mechanism of quenching and change of particle size gives rise to the binding force. Based on the FRET theory, the distances between potassium perfluorooctanesulfonate/sodium octanesulfonate and BSA are calculated and it is found that the fluorinated surfactant exhibits stronger interactions with proteins than the hydrogenated one, which is also proved by zeta potential and TEM.     

光谱法研究氨基己酸与牛血清白蛋白的相互作用

利用荧光光谱、紫外-可见吸收光谱及圆二色（CD）光谱研究了模拟生理条件下的氨基己酸(ACA)与牛血清白蛋白(BSA)的相互作用。 实验结果分析表明,氨基己酸对BSA的内源性荧光具有猝灭作用,属于动态猝灭过程。 计算了2种温度下ACA-BSA体系的结合常数、结合位点数及反应的热力学参数ΔG、ΔH和ΔS分别约为-21.00 kJ/mol、-0.64 kJ/mol和-72.00 kJ/(mol·K),由此推出了二者主要通过氢键和范德华力形成摩尔比为1∶1的复合物。 依据Forster非辐射能量转移理论求得二者之间的结合距离为2.3 nm。 位点取代实验指出氨基己酸主要结合在位点Site I。 CD光谱表明,氨基己酸诱导了BSA分子二级结构微变。     

Chiral protein scissors activated by light: recognition and protein photocleavage by a new pyrenyl probe

Strong chiral discrimination and site-selective photocleavage of two model proteins, lysozyme and bovine serum albumin (BSA), by new pyrenyl probes are reported here. The enantiomeric pyrenyl probes D-phenylalanine-1(1-pyrene)methylamide (PMA- D-Phe) and L-phenylalanine-1(1-pyrene)methylamide (PMA- l-Phe) were synthesized by coupling the carboxyl function of D-phenylalanine or L-phenylalanine with the amino group of 1(1-pyrene)methylamine. Binding affinities of the two enantiomers with the proteins were quantitated in absorption titrations. BSA indicated 10-fold selectivity for PMA- D-Phe, and the binding constants for the L- and D-enantiomers were 3.8 x 10(5) and 4.0 x 10(6) M(-1), respectively. Lysozyme, similarly, indicated a 6-fold preference for PMA- D-Phe with binding constants of 3.3 x 10 (5) and 2.0 x 10(6) M(-1) for the L- and D-isomers, respectively. Such strong chiral discrimination illustrates the key role of the chiral center of the probe (Phe) in the binding interactions. The enantiomers were tested to examine how the chiral discrimination for their binding influences reactivity toward protein photocleavage. Irradiation of the probe-protein complexes, at 342 nm in the presence of hexammine cobalt(III) chloride, resulted in the cleavage of the protein backbone. Photocleavage did not proceed in the dark or in the absence of the pyrenyl probes. Both enantiomers indicated low reactivity with BSA (<5% yield), while large photocleavage yields ( approximately 57%) have been noted with lysozyme. This lysozyme photocleavage yield is a significant improvement over previous reports. However, both enantiomers cleaved lysozyme at the same location between Trp108-Val109, despite the strong chiral selectivity for binding. H-atom abstraction from Trp 108, accessible from the active site cleft, could initiate the observed peptide bond cleavage.     

荧光光谱法研究一种噻吩并[2,3-d]嘧啶-4(3H)-酮衍生物与牛血清白蛋白的相互作用

用荧光光谱法研究了在生理条件下,3-对氯苯基-2-(4-叔丁基苯氧基)-3’-氧环己烷并噻吩并[2,3-d]嘧啶-4(3H)-酮(PTP)与牛血清白蛋白(BSA)的相互作用。实验表明,PTP主要以静态猝灭方式使BSA荧光强度显著降低。计算了不同温度下二者的结合常数和结合位点数,并根据热力学参数确定了PTP与BSA之间的作用力主要为氢键或范德华力。根据Frster非辐射能量转移机理,测定了PTP与BSA相互结合时的作用距离,并用同步荧光技术讨论了其衍生物对BSA构象的影响。     

四种黄酮类化合物与牛血清白蛋白相互作用的光谱分析

在人体生理pH条件下,利用紫外吸收光谱和荧光光谱研究了槲皮素(QUE)、大豆甙元(DAI)、4′,7-二甲氧基-3′-异黄酮磺酸钠(DISS)和3′-大豆甙元磺酸钠(DSS)四种黄酮类化合物与牛血清白蛋白(BSA)的相互作用,结合反应机理对其进行了初步探讨;并计算了结合位点数和结合常数.紫外吸收光谱分析结果表明,在pH=7.4条件下,黄酮类化合物中疏水性的苯环与BSA疏水腔中的氨基酸残基发生作用,从而导致药物分子的吸收峰红移,用Scatchard拟合法可求得DAI及DSS与BSA的结合常数.荧光光谱分析结果表明,BSA对DAI、DISS和DSS均有明显的敏化增强效应,计算得到的增强速率常数分别为1.39×1011,7.72×1011和1.93×1012L·s-1·mol-1,并可求得结合位点数和结合常数.     

Characterization of drug interactions with soluble beta-cyclodextrin by high-performance affinity chromatography

This study examined the use of an immobilized human serum albumin (HSA) column to study solution-phase reactions between drugs and beta-cyclodextrin (beta-CD). Chromatographic equations were developed to characterize the binding of chemicals to a soluble ligand (beta-CD) in the presence of an independent immobilized ligand (HSA). Situations considered included the presence of both a homogeneous and heterogeneous immobilized ligand, as well as complex interactions between the chemical of interest and soluble ligand. Three drugs (warfarin, tamoxifen, and phenytoin) were examined by this approach. This method involved injecting a small amount of each drug onto an HSA column in the presence of various concentrations of beta-CD in the mobile phase. By measuring the change in the drug's retention factor as the concentration of beta-CD was varied, it was possible to determine the stability constant between the injected drug and beta-CD. With this approach, warfarin and beta-CD were found to have 1:1 interactions with a stability constant of 5.2 x 10(2) M(-1) at 37 degrees C and pH 7.4, a result in close agreement with previous literature values. Tamoxifen and phenytoin were also found to have 1:1 interactions with beta-CD and had stability constants of 0.9-1.2 x 10(4) and 6-9 x 10(2) M(-1) respectively. With these latter solutes, the effects of secondary binding to the chromatographic support had to be considered. The theory and methods described in this report are not limited to these drugs and beta-CD but can be applied to other analytes and soluble ligands.     

葛根素及其衍生物与牛血清白蛋白相互作用研究

采用改造后的Atherton-Todd反应合成了葛根素的两种磷酰化异黄酮, 并应用荧光光谱法研究了葛根素及其磷酰化产物与牛血清白蛋白(BSA)的相互作用. 结果显示葛根素及其磷酰化产物均能与BSA发生相互作用, 但磷酰化产物与蛋白的结合作用相对较弱; 三个小分子对BSA荧光的猝灭是静态猝灭过程, 结合力以疏水作用力为主; 依据能量转移原理求得小分子与BSA间结合距离均小于7 nm. 通过比较葛根素及其磷酰化产物与BSA的相互作用, 初步探讨了三个小分子分子结构与其结合能力之间的联系, 并进一步考察了金属离子对结合反应的影响.     

Interaction of aspirin and vitamin C with bovine serum albumin

Vitamin C (L-ascorbic acid) has a major biological role as a natural antioxidant. Aspirin belongs to the nonsteroidal anti-inflammatory drugs and functions as an antioxidant via its ability to scavenge-OH radicals. Bovine serum albumin (BSA) is the major soluble protein constituent of the circulatory system and has many physiological functions including transport of a variety of compounds. In this report, the competitive binding of vitamin C and aspirin to bovine serum albumin has been studied using constant protein concentration and various drug concentrations at pH 7.2. FTIR and UV-Vis spectroscopic methods were used to analyze vitamin C and aspirin binding modes, the binding constants and the effects of drug complexation on BSA stability and conformation. Spectroscopic evidence showed that vitamin C and aspirin bind BSA via hydrophilic interactions (polypeptide and amine polar groups) with overall binding constants of K(vitamin C-BSA)=1.57×10(4)M(-1) and K(aspirin-BSA)=1.15×10(4)M(-1); assuming that there is one drug molecule per protein. The BSA secondary structure was altered with major decrease of α-helix from 64% (free protein) to 57% (BSA-vitamin C) and 54% (BSA-aspirin) and β-sheet from 15% (free protein) to 6-7% upon drug complexation, inducing a partial protein destabilization.