天竺葵素与人血清白蛋白相互作用的光谱学研究

采用荧光光谱、紫外吸收光谱和同步荧光光谱法研究了天竺葵素与人血清蛋白(HSA)的相互作用.结果表明:天竺葵素能使HSA发生内源荧光猝灭,属静态猝灭机理.25、30和37℃下,天竺葵素与HSA的静态猝灭速率常数分别为3.357×104,4.288×104和4.851×104L·mol-1,结合常数分别为3.24×104、...     

Fatty Acid Binding to Human Serum Albumin in Blood Serum Characterized by EPR Spectroscopy

One of the functions of Human Serum Albumin (HSA) is binding and transport of fatty acids. This ability could be altered by the presence of several blood components such as toxins or peptides – which in turn alters the functionality of the protein. We aim at characterizing HSA and its fatty acid binding in native serum environment. Native ligand binding and deviations from normal function can be monitored by electron paramagnetic resonance (EPR) spectroscopy using spin labeled fatty acids (FAs). Blood serum from healthy individuals is used to examine healthy HSA in its natural physiological conditions at different loading ratios of protein to FAs. Among the EPR spectroscopic parameters (like hyperfine coupling, line shape, rotational correlation time and population of different binding sites) the rotational correlation time is found to differ significantly between binding sites of the protein, especially at loading ratios of four FAs per HSA. Although differences are observed between individual samples, a general trend regarding the dynamics of healthy HSA at different loading ratios could be obtained and compared to a reference of purified commercially available HSA in buffer.     

碘的凝胶色谱测定方法

建立了碘折凝胶色谱测定方法。色谱条件为：Ｓｈｉｍ－ｐａｃｋ ＤＩＯＬ－１５０柱；流动相甲醇０．０１ｍｏｌ／Ｌ Ｈ３ＰＯ４（１０：９０）流速为１．２ｍＬ／ｍｉｎ；柱温为３５℃，检测波长为２２４ｎｍ；线性范围是０．０１０－１．０ｍｇ／Ｌ，相关系数为０．９９９３，检测限为０．００１ｍｇ／Ｌ，相对标准偏差为１．２％－４．６％；回收率为９１％－９８％。所建立的方法已用于食盐，尿，系带等样品的测试，均取得较好     

补骨脂素和异补骨脂素键合人血清白蛋白的比较

将互为同分异构体的两种植物药活性组分补骨脂素和异补骨脂素作为研究对象,利用荧光光谱、紫外光谱、圆二色谱及傅立叶变换红外光谱详细比较研究了这两种香豆素类化合物与人血清白蛋白(HSA)的键合作用.不同光谱的结果定性、定量地显示了HSA二级结构变化的程度.依据荧光滴定实验及Van′t Hoff公式求出了反应的热力学参数(ΔH和ΔS)的值.根据修正后的Stern-Volmer和Scatchard方程和荧光光谱数据分别求得不同温度(296,303,310及318 K)下药物与蛋白相互作用的结合常数及结合位点数;且根据F觟rster偶极-偶极能量转移理论,求得药物与HSA间的键合距离;利用竞争实验确定了药物在HSA上的键合位点为site II.从分子水平上揭示了这两种化合物与HSA相互作用的机制.     

蒽醌及黄酮类化合物与人血清白蛋白的结合反应研究

用荧光光谱法研究了大黄素，黄酮，栎精。柚皮甙，黄芩甙和龙胆苦甙等化合物与人血清白蛋白（ＨＳＡ）的结合反应，发现除龙胆苦甙外，其余化合物对ＨＳＡ的荧光有很强的猝平作用。从荧光猝灭结果求得了它们与ＨＳＡ的结合常数，表明这些化合物与ＨＳＡ的结合能力随其所含极性基团的增多而减弱。根据Ｆｏｒｓｔｅｒ非辐射能量转移机理，求出第一结合部位与ＨＳＡ分子中２１４－色氨酸残基的距离，由此提出了形成复合物的结构模型，并     

Study of the Binding of Herbacetin to Bovine Serum Albumin by Fluorescence Spectroscopy

In this paper, the interaction between herbacetin and BSA was investigated by fluorescence and three-dimensional fluorescence spectroscopy under simulated physiological conditions. It was proved that the fluorescence quenching of BSA by herbacetin was mainly the result of the formation of a herbacetin–BSA complex. The modified Stern–Volmer quenching constant and the corresponding thermodynamic parameters ΔH ⁰, ΔG ⁰ and ΔS ⁰ were calculated at different temperatures. The results indicated that electrostatic interactions were the predominant intermolecular forces in stabilizing the complex. The distance r=3.23 nm between the donor (BSA) and acceptor (herbacetin) was obtained according to Förster’s nonradioactive energy transfer theory. The synchronous fluorescence and three-dimensional fluorescence spectra results showed that the hydrophobity of amino acid residues increased in the presence of herbacetin. These results revealed that the microenvironment and conformation of BSA changed during the binding reaction.     

A Thermodynamic Study on the Binding of Human Serum Albumin with Lanthanum Ion

Thermodynamics of the interaction between lanthanum(III) ion, La³⁺, and human serum albumin (HSA), was investigated at pH 7.0 and 300 K in Tris‐HCl buffer by isothermal titration calorimetry. A solvation model was used to reproduce the enthalpies of HSA interaction with La³⁺. The solvation parameters recovered from our new model were attributed to the structural change of HSA and its biological activity. The interaction of HSA with La³⁺ showed a set of two binding sites with negative cooperativity.     

Unusual Binding of a Potential Biomarker with Human Serum Albumin

This study investigates the specific binding of a potential biomarker, [2,2′‐bipyridyl]‐3,3′‐diol (BP(OH)₂), with human serum albumin (HSA). The binding of BP(OH)₂ at the two primary drug‐binding sites on HSA (Sudlow′s sites I and II) is explored by a competitive‐binding study and monitored by considering the green‐light emission from its diketo tautomer. Warfarin is used as a marker for site I and dansyl‐L ‐proline (DP) as a competitor for site II. Steady‐state and time‐resolved fluorescence measurements affirm that neither of Sudlow′s sites is the binding locus of BP(OH)₂. To gain an idea regarding the probable binding site of BP(OH)₂, we perform molecular‐docking studies, which reveal a close proximity of the probe to Trp‐214 in subdomain IIA of HSA. Confirmation of this contention is achieved by studying the quenching of the fluorescence of Trp‐214 in the presence of BP(OH)₂. Moreover, static quenching seems to be responsible for the depletion of the fluorescence of Trp‐214, as manifested by the invariance of the intrinsic fluorescence lifetime of Trp‐214, as a function of the concentration of BP(OH)₂. Based on displacement and quenching studies, supported by molecular docking, we propose that BP(OH)₂ binds in a cleft that separates subdomains IIIA and IIB, which is in close proximity to Trp‐214.     

甲磺酸培氟沙星与人血清白蛋白之间结合模式的研究

采用实验和计算的方法研究了甲磺酸培氟沙星与人血清白蛋白之间的结合作用.荧光法测得甲磺酸培氟沙星与人血清白蛋白形成一种类型的复合物,结合常数为1.7×10⁵ L&;#8226;mol^－1,有1.05个平均结合位点；微量热法测得该药物-蛋白结合过程中焓变为1.03 kJ&;#8226;mol^－1,熵变为101.28 J&;#8226;K^－1&;#8226;mol^－1,反应为熵驱动.用分子对接的方法预测了甲磺酸培氟沙星与人血清白蛋白的结合模式.计算表明,甲磺酸培氟沙星可结合在人血清白蛋白的两个药物结合位点,疏水作用即熵效应在药物与蛋白的结合中起重要作用,预测的结合自由能和实验值基本一致.     

Chromatographic Framework to Determine the Memantine Binding Mechanism on Human Serum Albumin Surface

In this work, the interaction of memantine with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. The determination of the enthalpy change at different pH values suggested that the protonated group in the memantine–HSA complex exhibits a heat protonation with a magnitude around 65 kJ mol^?1. This value agrees with the protonation of a guanidinium group, and confirmed that an arginine group may become protonated in the memantine–HSA complex formation. The thermodynamic data showed that memantine–HSA binding, for low temperature (<293 K), is dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge–charge interactions contribute to the memantine–HSA complex formation. Above 293 K, the thermodynamic data ΔH and ΔS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface. The temperature dependence of the free energy of binding is weak because of the enthalpy–entropy compensation caused by a large heat capacity change, ΔC _p = ? 3.79 kJ mol^?1 K^?1 at pH = 7. These results were used to determine the potential binding site of this drug on HSA.     

Competitive Binding of Drugs to the Multiple Binding Sites on Human Serum Albumin: A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.This revised version was published online in November 2005 with corrections to the Cover Date.     

Conformation and Thermodynamic Properties of the Binding of Vitamin C to Human Serum Albumin

The binding of vitamin C, L-ascorbic acid (AsA), with human serum albumin (HSA) was investigated by various spectroscopic techniques under simulated physiological conditions. The fluorescence quenching constants (K _SV) at four different temperatures (292, 298, 304, and 310 K) were obtained. The thermodynamic parameters ΔH ^∘ and ΔS ^∘ were calculated to be 6.02 kJ⋅mol⁻¹ and 84.55 J⋅mol⁻¹⋅K⁻¹ using the van’t Hoff equation. Additional experiments to determine the stoichiometry (n) were carried out using isothermal titration calorimetry (ITC) and cyclic voltammetry (CV). The distance, r, between AsA and the tryptophan residues of HSA was calculated to be 3.7 nm according to F?rster’s non-radiation energy transfer theory. The effect of AsA on the conformation of HSA was studied by means of three dimensional fluorescence spectra and CD spectra. The results indicate that the presence of AsA resulted in a slight change of the HSA secondary structure. The effect of common ions on the binding of AsA to HSA was also examined.     

芘与人血清白蛋白和牛血清白蛋白结合位点微环境极性的差异

利用芘(Pyr)的微环境极性探针性质, 采用稳态荧光光谱、 荧光共振能量转移技术结合分子对接法, 对比分析了Pyr分别与人血清白蛋白(HSA)和牛血清白蛋白(BSA)作用机制的差异. 结果表明, HSA和BSA中Pyr的I₁/I₃平均值分别为1.36和0.92; Pyr与HSA和BSA的结合常数分别为1.86×10⁷和1.71×10⁵ L/mol; Pyr与HSA和BSA中色氨酸残基表观距离分别为2.37和2.34 nm. Pyr在HSA和BSA中不同的结合位点位于ⅠB子域和ⅠA子域, 其结合位点周围氨基酸残基的极性是影响Pyr I₁/I₃值的主要原因之一. 实验证实Pyr与HSA和BSA结合作用位点处的微环境极性存在差异.     

化学改性纤维素亲和膜色谱用作人血清白蛋白中杂质的去除

用环氧法对纤维素微孔滤膜进行了化学改性，并在经改性的膜上共价结合了活性红染料配基Ｋ－２ＢＰ，设计并制作了特殊的膜分离器，利用所制备的亲和膜分离器成功地去除了人血清白蛋白中的杂质。     

Use of Frontal Chromatography to Measure the Binding Interaction of Berberine Chloride with Bovine Serum Albumin

There is much interest in the interactions between the active constituents of traditional Chinese medicine and biomolecules. By use of frontal analysis on an affinity column we have examined the binding interaction of berberine chloride (BC), a major active constituent of coptis, with bovine serum albumin (BSA) in 40 mM phosphate buffer, pH 7.0. Adsorption of BC on immobilized BSA was in accordance with the Langmuir isotherm, suggesting BC is binding to a single type of site on the immobilized BSA. The binding constant was 4.79 × 10⁴ L mol⁻¹ at 30 °C, less than the value of 6.61 × 10⁴ L mol⁻¹ obtained by fluorescence spectroscopy under the same buffer and temperature conditions. The effects of temperature on the retention, binding constant, and active binding sites, and on the percentage protein binding of BC, were also investigated. Thermodynamic measurements indicated that the increase in entropy was an important process promoting the interaction between BC and BSA.

     

Use of Frontal Chromatography to Measure the Binding Interaction of Berberine Chloride with Bovine Serum Albumin

There is much interest in the interactions between the active constituents of traditional Chinese medicine and biomolecules. By use of frontal analysis on an affinity column we have examined the binding interaction of berberine chloride (BC), a major active constituent of coptis, with bovine serum albumin (BSA) in 40 mM phosphate buffer, pH 7.0. Adsorption of BC on immobilized BSA was in accordance with the Langmuir isotherm, suggesting BC is binding to a single type of site on the immobilized BSA. The binding constant was 4.79 × 10⁴ L mol^?1 at 30 °C, less than the value of 6.61 × 10⁴ L mol^?1 obtained by fluorescence spectroscopy under the same buffer and temperature conditions. The effects of temperature on the retention, binding constant, and active binding sites, and on the percentage protein binding of BC, were also investigated. Thermodynamic measurements indicated that the increase in entropy was an important process promoting the interaction between BC and BSA.     

一种新型三氮唑化合物的光物理性质及其与血清白蛋白的键合研究

1-(1-萘基)-5-苯基-1H-1,2,3-三唑(1-(naphthalen-1-yl)-5-phenyl-1H-1,2,3-triazole, 简称NPTA)是一种新合成的三氮唑化合物, 研究了其光谱特征及其生物活性. 利用PM3半经验方法的Polak-Ribiere共轭梯度法得到了优化的NPTA分子结构, 进而用分子对接显示与人血清白蛋白(HSA)的键合模式及位点, 再利用紫外光谱、多种荧光光谱法等技术表征了NPTA及其键合HSA的光谱性质. 结果表明: 共轭多烯π键的存在使得NPTA呈现特征的荧光与紫外光谱. 分子模拟的结果表明NPTA可以嵌于HSA分子的疏水腔内, 并与精氨酸Arg222形成四个氢键; 位点竞争实验确定了NPTA在HSA亚结构域的位点II位发生作用. 二维及三维荧光光谱显示NPTA可以猝灭HSA的内源荧光, 使其构象发生变化; 紫外、同步荧光扫描证实NPTA主要猝灭了HSA色氨酸残基的荧光, 并影响了HSA的微环境; 较小的各向异性值说明NPTA与HSA结合后生成的配合物弛豫时间较短, 结合的较松. 荧光滴定法求得不同温度下(299, 309和319 K) NPTA与HSA作用的键合常数, 键合模式为典型的疏水作用, 与分子模拟的结果相一致. 另外, 测定了NPTA与键合HSA相关的几种物理化学参数.     

茜素红S-牛血清白蛋白配合物的凝胶层析分离制备及紫外可见吸收光谱研究

用葡聚糖凝胶层析分离制备牛血清蛋白(BSA)-茜素红S(ARS)配合物。在420和530 nm处用紫外可见吸收光谱法同时测定含有BSA-ARS配合物和茜素红S的联立方程为:A420=4.89×103cARS 3.06×104cBSA-ARS,A530=4.60×102cARS 2.29×104cBSA-ARS;正交试验选择了合适的分离条件:柱直径1.0 cm,柱长30.0 cm,凝胶用量1.3 g,最佳进样浓度为ARS:5×10-3mol/L、BSA:1.49×10-4mol/L,进样体积1.5 mL,洗脱流速0.33 mL/min,分离度为1.25;测定了纯BSA-ARS配合物紫外吸收光谱,最大吸收峰在530 nm。     

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single‐molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 ( C153 , neutral) and rhodamine 6G ( R6G , cationic) were chosen. For both the dyes, a major (ca. 96‐98 %) and minor (ca. 3 %) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ_off and τ_on) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst‐integrated lifetime analysis. Distributions of lifetime histograms for both the probes ( C153 and R6G ) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153 . However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.