Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 mol L^–1). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Characterization of Interaction Between Raltitrexed and Bovine Serum Albumin by Optical Spectroscopic Techniques

The interaction of raltitrexed(RTX) with bovine serum albumin(BSA) was investigated by steady state/lifetime fluorescence spectroscopy and circular dichroism(CD) spectroscopy under the simulative physiological conditions. The results of fluorescence titration reveal that RTX could strongly quench the intrinsic fluorescence of BSA via a static quenching procedure. The obtained binding constant K_A of RTX with BSA was 478630 and 44259 L/mol at 298 and 310 K, respectively. According to van’t Hoff equation, the thermodynamic parameters ΔH, ΔG and ΔS were calculated, indicating that hydrophobic forces were the predominant intermolecular forces in stabilizing the complex. The binding process was a spontaneous process, in which Gibbs free energy change was negative. According to Förster’s non-radioactive energy transfer theory, the distance r between donor(BSA) and acceptor(RTX) was 3.82 nm, suggesting that the energy transfer from BSA to RTX occurred with high probability. Displacement experiment and the number of binding sites calculation confirmed that RTX could bind to the site-I of BSA. Furthermore, the effects of pH and some metal ions on the interaction of RTX with BSA were also investigated. The results of synchronous fluorescence and CD spectra show that the RTX-BSA binding induced conformational changes in BSA.     

Studies on the binding of nevadensin to human serum albumin by molecular spectroscopy and modeling

The binding of nevadensin to human serum albumin (HSA) in aqueous solution was investigated for the first time by molecular spectroscopy and modeling at pH 7.4. Spectrophotometric observations are rationalized in terms of a static quenching process and binding constant (K_a, K_b) and the number of binding sites (n ≈ 1) were evaluated by fluorescence quenching methods. Thermodynamic data showed that nevadensin was included in the hydrophobic cavity of HSA mainly via hydrophobic interactions. The value of 3.09 nm for the distance r between the donor (HSA) and acceptor (nevadensin) was derived from the fluorescence resonance energy transfer. Spectrophotometric techniques were also applied to investigate the structural information of HSA molecules on the binding of nevadensin and the results showed that the binding of nevadensin to HSA did not change significantly molecular conformation of HSA in our experimental conditions. Furthermore, the study of molecular modeling also indicated that nevadensin could strongly bind to the site I (subdomain IIA) of HSA mainly by a hydrophobic interaction and there are hydrogen bond interactions between nevadensin and the residues Arg-218, Arg-222, Lys-195, and Asp-451. As compared to the other flavonoids, the flavonoids containing methoxy groups which are in aromatic rings can bind to HSA with higher affinity.     

大黄酸铜(Ⅱ)配合物与血清白蛋白的相互作用

采用荧光光谱法和紫外光谱法研究了大黄酸铜配合物与牛血清白蛋白之间的相互作用.大黄酸铜配合物能显著猝灭牛血清白蛋白的内源荧光并以静态猝灭为主;计算了298 K和309 K温度下结合常数、结合位点,根据热力学参数判断大黄酸铜配合物与牛血清白蛋白之间具有较强的疏水作用力;依据F?rster的偶极-偶极非辐射能量转移理论,计算出大黄酸铜在蛋白质中结合位置与色氨酸残基间的距离为3.21 nm, 表明大黄酸铜的部分片段能够插入蛋白质分子内部;用同步荧光光谱和圆二色光谱技术探讨了大黄酸铜对牛血清白蛋白构象的影响.     

Ratiometric Fluorescent Probe Based on Novel Red-emission BODIPY for Determination of Bovine Serum Albumin

A novel red-emission boron-dipyrromethene（BODIPY） dye with a pyrrole ring was synthesized simply via one-pot reaction. The spectral properties of it were investigated under the conditions of different solvents. The results show that the as-prepared BODIPY dye is extremely sensitive to solvent polarity, and the fluorescent emission enhances with the decrease of solvent polarity. In aqueous buffer, the addition of bovine serum albumin leads to a ratiometric change in absorption spectra with an association constant of 1.16×10^6 L/mol. Meanwhile, the fluorescence emission increases greatly at 622 nm but changes slightly at 575 nm. The response time is very short（less than 3 min）, and the changes of color can be noticed by naked eyes. Bovine serum albumin can be detected by this ratiometric fluorescence probe, but other proteins or enzymes cannot be detected by this method, which indicates that this novel dye has high selectivity towards bovine serum albumin. The reason is that bovine serum albumin has suitable hydro- phobic cavities for binding with the dye. In addition, the dye molecule can penetrate cell membrane easily and make a fast fluorescent stain, which makes it a potential probe for living-cell fluorescence imaging.     

抗肿瘤药物替加氟与牛血清白蛋白相互作用的热化学研究

在298.15 K下, 以等温滴定微量热(ITC)实验数据为依据,根据合理假设和Langmuir结合理论, 应用非线性最小方差拟合方法测定了抗肿瘤药物替加氟(Tegafur)与牛血清白蛋白(BSA)结合过程热力学性质的改变. 研究结果表明, 牛血清白蛋白与替加氟相互作用存在两类结合位点: (1) N=52.00±0.12, K=(9.83±0.13)×104 L/mol, ΔH=(30.10±0.17) kJ/mol>0, ΔS=(196.00±0.65) J/(mol·K)>0, ΔG=(-28.50±0.66) kJ/mol<0, 表现为熵驱动过程, 疏水相互作用为过程的主要推动力; (2) N=86.00±0.14, K=(9.35±0.13)×104 L/mol, ΔH=(-19.80±0.17) kJ/mol<0, ΔS=(28.30±0.50) J/(mol·K)>0, ΔG=(-28.40±0.43) kJ/mol<0, 表现为焓-熵协同过程, 氢键和静电相互作用为过程的主要推动力. 圆二色谱(CD)分析结果表明, 抗肿瘤药物替加氟诱导蛋白质(BSA)二级结构单元的相对含量发生了一定程度的变化.     

Study of the effect of Cal-Red on the secondary structure of human serum albumin by spectroscopic techniques

The effect of Cal-Red on the structure of human serum albumin (HSA) was studied using Resonance light scattering (RLS), Fourier transformed Infrared (FT-IR) and Circular dichroism (CD) spectroscopic methods. The RLS spectroscopic results show that the RLS intensity of HSA was significantly increased in the presence of Cal-Red. The binding parameters of HSA with Cal-Red were studied at different temperatures of 289, 299, 309 and 319 K at pH 4.1. It is indicated by the Scatchard plots that the binding constant K decreased from 4.03 × 10⁸ to 7.59 × 10⁷ l/mol and the maximum binding number N decreased from 215 to 152 with increasing the temperature, respectively. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The enthalpy change ΔH⁰, the free energy change ΔG⁰ and the entropy change ΔS⁰ of 289 K were calculated to be −42.75 kJ/mol, −47.56 kJ/mol and 16.66 J/mol K, respectively. The alterations of protein secondary structure in the presence of Cal-Red in aqueous solution were quantitatively calculated from FT-IR and CD spectroscopy with reductions of -helices content about 5%, β-turn from 10% to 2% and with increases of β-sheet from 38% to 51%.     

Determination of serum albumin in the presence of poly(diallyldimethylammonium chloride) by resonance light scattering technique

By means of the resonance light scattering (RLS) technique, a new method was developed to determine the bovine serum albumin (BSA) and human serum albumin (HSA) by the interaction of serum albumin with poly(diallyldimethylammonium chloride) (PDDA). At Tris-NaOH buffer solution, the RLS intensity of serum albumin at the wavelength 320, 550 and 590 nm was obviously enhanced in the presence of PDDA. The influences of some experimental factors, including incubation time, addition sequence of reagents, pH value, concentration of PDDA and foreign substances, on the enhancement of the RLS intensity were examined. The optimum conditions of the experiment were selected. Under the selected experimental condition, the enhanced RLS intensities were directly proportional to the concentrations in the range of (0.0250-2.75)x10(-6) mol/L for BSA and (0.0235-1.17)x10(-6) mol/L for HSA. The detection limits (S/N=3) were 8.40x10(-9) mol/L for BSA and 7.39x10(-9) mol/L for HSA. The synthetic samples were analysed and the results obtained were satisfactory.     

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.     

Characterization of peak purity by fast multiscan circular dichroism detection

Summary A method has been developed to detect inhomogeneity of apparently homogeneous peaks of very similar analytes. The method utilizes the rapid scan feature of state-of-the-art spectrometers/detectors that allow the recording of up to 30 spectra in a single chromatographic peak. Sensitivity and selectivity are enhanced by chiroptical/optical detection. Thus, identification of “front” and “rear” components of the peak can be carried out. The method is exemplified by mixtures of codeine, hydrocodone and oxycodone as analytes. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Studies of (3-mercaptopropyl)trimethoxylsilane and bis(trimethoxysilyl)ethane sol–gel coating on copper and aluminum

Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern–Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10⁴ L mol⁻¹ at 298 K. The thermodynamic parameters, enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were −76.5 kJ mol⁻¹ and −173.4 J mol⁻¹ K⁻¹ according to the van’t Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV–vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.     

维生素C、维生素B2和葡萄糖对杜鹃素与BSA结合的扰动

在生理酸度条件下(pH 7.4),采用荧光、紫外、红外及圆二色谱等方法,研究了3种典型营养物质维生素C(VC)、维生素B2( VB2)和葡萄糖(Glu)分别对植物活性成分杜鹃素与牛血清白蛋白(BSA)结合的扰动.计算了3种营养物质分别存在时BSA -杜鹃素体系的猝灭常数(Ksv)、结合常数(Ka)、结合位点数(n)及结...     

5-氟尿嘧啶与牛血清白蛋白的相互作用

根据合理的假设和Langmuir结合理论, 在298.15 K下, 以等温滴定微量热(ITC)实验数据为依据, 应用非线性最小方差拟合方法确定了抗肿瘤药物配5-氟尿嘧啶(5-FU)与牛血清白蛋白(BSA)相互作用的热力学性质的改变. 研究结果表明, 牛血清白蛋白(BSA)与5-氟尿嘧啶相互作用存在两类结合位点. 第一类结合, N=(54.0 0.3), ⊿H⁰=(30.0±0.4) kJ·mol^-1 (吸热), ⊿S⁰=(196.0±2.6) J·mol-1·K-1(熵增), ⊿G⁰=(-28.4±0.3) kJ·mol^-1; 第二类结合, N=(77.0±0.4), ⊿H⁰=(-20.0±0.4) kJ·mol^-1 (放热), ⊿S⁰=(28.6±0.3) J·mol^-1·K^-1 (熵增), ⊿G⁰=(-28.5±0.2) kJ·mol^-1. 结合体系的圆二色谱(CD)分析结果说明, 抗肿瘤药物5-氟尿嘧啶与BSA的相互作用诱导蛋白质(BSA)二级结构单元的相对含量发生了一定程度的变化.     

利福布汀与人血清白蛋白相互作用的光谱研究

用荧光光谱法和圆二色谱法研究了利福布汀(RB)与人血清白蛋白(HSA)的相互作用. 结果表明, RB与HSA之间的相互作用主要是疏水作用, 作用机制是静态猝灭与动态猝灭的结合. 其结合常数(Ka)在106数量级, 说明RB和HSA有很强的结合. 此外, 探讨了金属离子(Cu2+, Zn2+, Mg2+ 和Ca2+)对RB与HSA结合常数的影响. 同步荧光光谱和圆二色谱数据表明, RB可导致HSA的构象改变.     

大黄酸锌配合物与牛血清白蛋白相互作用的研究

采用荧光猝灭光谱、同步荧光光谱、紫外光谱和圆二色光谱,研究了大黄酸锌配合物(Rhein-Zn)与牛血清白蛋白(BSA)之间的相互作用.结果表明,Rhein-Zn能显著猝灭BSA的内源荧光并以静态猝灭为主;Rhein-Zn与BSA结合常数分别为1.3×107 (22 ℃)、1.6×106 (27 ℃)和1.0×105 L...     

Study of nobiletin binding to bovine serum albumin by capillary electrophoresis–frontal analysis and circular dichroism

A very recent epidemiological study provided strong support for nobiletin (NOB) as a potential candidate chemopreventive agent against cancer. From the pharmacology point of view, drug–protein interactions are determining factors in therapeutic, pharmacodynamic and toxicological drug properties. In this work, for the first time, detection of NOB at near‐physiological conditions was accomplished by means of capillary electrophoresis–frontal analysis (CE‐FA), and then the binding constants of NOB with bovine serum albumin (BSA) at the same conditions were determined. Complexation of NOB–BSA led to a decrease of the height for free NOB with increasing concentration of BSA. These results revealed the presence of a single class of binding site on BSA, and provided the binding constant of 10³/m , showing the strong affinity of NOB for BSA. Furthermore, circular dichroism spectra showed that, when the molar ratio of NOB to BSA was up to 2:1, NOB did not affect the overall protein conformation significantly and the protein thus retained a native‐like structure. These results may provide important information for preclinical studies of nobiletin in pharmaceutical research. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of L-tryptophan-serum albumin binding by high-performance liquid chromatography

Summary Two high-performance liquid chromatography (HPLC) techniques were developed for the determination of binding constants in the interaction of serum albumin with L-tryptophan: internal calibration and external calibration. The results obtained were compared with those obtained by the classical method of equilibrium dialysis and by gel filtration. While all the methods are equally reliable, the internal and external calibration techniques seem to be superior in their simplicity, speed and convenience.     

核黄素与牛血清白蛋白相互作用的荧光光谱研究

采用荧光猝灭光谱、同步荧光光谱研究了核黄素与牛血清白蛋白(BSA)相互作用的光谱行为。结果发现,在温度为293 K和310 K时核黄素与BSA的结合常数(Kb)分别为4.879×105L.mol-1和1.880×105L.mol-1,结合热力学方程计算得到了对应温度下的热力学参数。结果表明核黄素对BSA有较强的荧光猝灭作用,其荧光猝灭过程属于动态猝灭机制,二者主要靠疏水作用力结合。采用同步荧光光谱探讨了核黄素对BSA构象的影响。     

刚果红与血清白蛋白相互作用的极谱分析

在pH4．7HAc-NaAc缓冲溶液中，刚果红与蛋白质(BSA或HSA)作用形成络合物，使刚果红-0．35V(vs SCE)的还原峰电流下降，峰电流的降低值同所加的BSA或HSA质量浓度在一定范围内呈线性关系；BSA和HSA的线性范围分别为0．5～12mg／L和0．5～11mg／L，检出限分别为0．25和0．20mg／L；运用该法测定了人血清白蛋白样品，结果令人满意。