α-硫辛酸与牛血清白蛋白的相互作用

利用荧光光谱和紫外-可见吸收光谱研究了在缓冲溶液中不同温度下α-硫辛酸(ALA)与牛血清白蛋白(BSA)的相互作用。结果表明,ALA对BSA的内源荧光猝灭为静态猝灭过程,猝灭常数KSV分别为4.65×103L/mol(26℃)和4.46×103L/mol(37℃)。依据Frster非辐射能量转移机制,得到给体(BSA)-受体(ALA)间的结合距离r=2.90 nm,能量转移效率E=5%。测定了该反应在不同温度下的结合常数KA=4.31×103L/mol(26℃),4.27×103L/mol(37℃),以摩尔比1∶1结合。根据不同温度下的结合常数确定了相互作用过程的热力学参数,并根据热力学参数确定了ALA与BSA之间作用力主要是静电作用。     

毛细管电泳电化学淌度移动法测定利尿剂和牛血清白蛋白结合常数

采用毛细管电泳淌度移动法研究了利尿剂氢氯噻嗪(HCT),布美他尼(BMTN)与牛血清白蛋白(BSA)的结合常数.使用未涂层的毛细管柱(70 cm×25 μm i.d.),中性H3BO3-Na2B4O7-NaCl(Palitzsch, pH 7.4)缓冲溶液,在800 mV工作电位,分离电压9 kV和电动进样9 kV×8 s的条件下,测得HCT,BMTN与BSA的结合常数分别为2.45×104 L/moL,1.19×104 L/moL.得到的结合常数值与经典紫外分光光度法测定的值相近.实验表明,本方法可以作为研究药物与蛋白相互作用的一种快速、简便的检测方法.     

甲基紫与牛血清白蛋白作用的荧光光谱研究

在pH 7.40的Tris-HC1缓冲溶液体系中,采用荧光光谱法和同步荧光光谱法研究了甲基紫(MV)与牛血清白蛋白(BSA)相互作用.结果表明MV与BSA相互作用两者存在一个结合位点,结合常数(KA)为7.628×103 L/mol,MV与BSA主要发生疏水作用,反应是一个吸热、熵增的自发过程.     

卡维地洛的电化学测定及与牛血清白蛋白相互作用的研究

建立了测定卡维地洛(CAR)新的电化学方法.结合紫外、红外光谱分析,研究了CAR与牛血清白蛋白(BSA)的相互作用.实验发现,在pH4.0Britton-Robinson(B-R)缓冲溶液中,CAR在碳糊电极上产生3个不可逆的氧化峰.以0.92V处的氧化峰为研究对象,结果发现峰电流Ipa,1与CAR浓度在2.45×10-5～1.19×10-3mol/L范围呈良好的线性关系,CAR的检出限为5.6×10-6mol/L.当BSA加入CAR溶液后,CAR峰电流降低,氧化峰电流的降低值△Ipa,1与BSA的浓度在2.92×10-7～1.09×10-5mol/L范围内呈良好线性关系,BSA的检出限为4.1×10-8mol/L.电化学结果表明,CAR与BSA之间形成1∶1的结合物,结合常数为3.14×106L/mol.紫外光谱表明CAR的加入使BSA的吸收峰发生红移且有增色效应.红外光谱表明CAR与BSA分子中氨基酸残基的硫及氮原子形成键合作用.     

盐酸胺碘酮的电化学测定及与牛血清白蛋白的相互作用

建立了测定盐酸胺碘酮(Am)的新的电化学分析方法。采用电化学方法结合紫外、红外光谱分析,研究了Am与牛血清白蛋白(BSA)的相互作用。实验发现,在pH=5.0的B-R缓冲溶液中,Am在0.87V处有一灵敏的氧化峰,其氧化峰电流Ipa与Am的浓度在1.8×10-7～6.7×10-5 mol/L范围呈良好的线性关系,检出限(S/N=3)为6.0×10-8 mol/L。当BSA加入Am溶液后,Am的氧化峰电流降低,其氧化峰电流的降低值△Ipa与BSA的浓度在2.4×10-7～2.2×10-5 mol/L范围内呈良好线性关系,BSA的检出限(S/N=3)为9.0×10–8 mol/L。电化学研究表明,Am与BSA之间形成1∶1的结合物,结合常数为9.9×106 L/mol。紫外光谱表明Am的加入使BSA的吸收峰发生红移且有增色效应。傅立叶红外光谱表明Am与BSA分子中氨基酸残基的硫及氮原子形成键合作用。     

甲氧氯普胺的测定及与牛血清白蛋白相互作用的研究

建立了多壁碳纳米管糊电极测定盐酸甲氧氯普胺(MCPM)的电化学分析方法,研究了MCPM与牛血清白蛋白(BSA)相互作用的电化学行为。在pH2.2的Britton-Robinson(B-R)缓冲溶液中,MCPM在1.084V处出现一灵敏的氧化峰。电化学测定MCPM的线性范围为0.17～10.3mg/L,检出限为0.03 mg/L。对5.18mg/L的MCPM进行11次平行测定,相对标准偏差为2.4%。当BSA加入到MCPM溶液中,MCPM的氧化峰电流明显降低。其降低值(ΔIp)与BSA的浓度在5.00×10-7～1.67×10-5mol/L范围内呈良好线性关系,检出限为2.0×10-7mol/L。MCPM与BSA相互作用的结合常数为1.05×105L/mol。     

亲和毛细管电泳法测定牛血清白蛋白和加替沙星的结合常数

利用亲和毛细管电泳法对牛血清白蛋白(BSA)与加替沙星(GT)间的结合反应及其相互作用做了初步探索,并应用淌度比(M)作为指标测定了两者的结合常数。以20 mmol/L pH 7.4的磷酸盐缓冲液作为运行缓冲液,分别以GT和BSA作为添加剂,另一组分为进样样品,内标为二甲基甲酰胺,于214 nm波长下检测。两种测定条件下得到的结合常数分别为4.4×104 L/mol和4.2×104 L/mol,与传统的荧光淬灭法测得的结果基本一致。该方法具有简单、高效的优点。     

蛋白质与聚乙二醇之间相互作用的光谱法研究

通过共振散射光谱(RLS)、二维共振散射光谱(2D-RLS)及荧光光谱研究了牛血清白蛋白(BSA)与聚乙二醇(PEG)在溶液中的相互作用。PEG和BSA之间存在着氢键作用及疏水作用,其中氢键作用是主要的。在相同条件下,基于RLS方法测得的PEG与BSA之间的结合常数为9.56×103L/mol,而基于荧光光谱方法测得的PEG与BSA之间的结合常数为6.21×103L/mol。两种方法此外所测得的实验数据比较接近,表明RLS方法可普遍用来分析蛋白质与水溶性聚合物之间相互作用。     

毛细管电泳法研究牛血清白蛋白与盐酸异丙肾上腺素的相互作用

本研究分别采用区段-区段动力学毛细管电泳法(Plug-Plug Kinetic Capillary Electrophoresis,ppKCE),及以药物为添加剂的亲和毛细管电泳法(Affinity Capillary Electrophoresis,ACE)对盐酸异丙肾上腺素(Hydrochloric Acid Isoproterenol,HAI)与牛血清白蛋白(Bovine Serum Albumin,BSA)的结合作用进行研究。实验结果显示,ppKCE法可同时测得HAI与BSA相互作用体系的结合速率常数kon和离解速率常数koff分别为163.60L·mol-1·s-1、3.50×10-2±0.95×10-2s-1(n=3)。ppKCE法和ACE法测得HAI与BSA的结合常数Kb分别为4.67×103 L·mol-1、6.02×103 L·mol-1,两种方法所得结果能够较好吻合。证明毛细管电泳法在药物-蛋白相互作用研究领域的可靠性,可用于测定药物与蛋白的相互作用,从而用于新药筛选研究。     

荧光光谱法研究染料木苷与牛血清白蛋白的相互作用

采用荧光光谱法,在pH=7.4的磷酸盐缓冲溶液(PBS)中研究了染料木苷(Genistin,Ge)与牛血清白蛋白(BSA)的作用方式及作用机理.结果表明,Ge可静态猝灭BSA的内源荧光,二者之间的结合位点数为1.4,温度对结合常数影响不大,300K、306 K、310 K和313 K的结合常数平均为4.31×104 L...     

Bovine serum albumin-mediated enantiodifferentiating photocyclodimerization of 2-anthracenecarboxylate

Enantiodifferentiating photocyclodimerization of 2-anthracenecarboxyalate (AC) was performed at 25 degrees C in aqueous buffer solution (pH 7) in the presence of bovine-serum albumin (BSA) to afford four [4 + 4] cyclodimers, i.e., anti- and syn-head-to-tail (HT) (1 and 2) and anti- and syn-head-to-head (HH) dimers (3 and 4), of which only 2 and 3 are chiral. We found that (1) BSA possesses four sets of binding sites for AC of different affinities, stoichiometries, and chiral environment for photoreaction, which bind 1, 3, 2, and 3 AC molecules with binding constants of 5.3 x 107, 1.3 x 105, 1.4 x 104, and 3.0 x 103 M-1, respectively, (2) the regioselectivity of photodimerization is switched from HT to HH by adding BSA (the HH/HT ratio varies from 0.28 to 4.3), (3) BSA-mediated photodimerization of AC affords optically active products 2 and 3 of up to 29% and 41% ee, respectively. It is emphasized that the selective excitation of bound substrate, utilizing the spectral shift upon complexation with BSA, is not a prerequisite for efficient photochirogenesis using biomolecules.     

Interaction between fluoroquinolones and bovine serum albumin studied by affinity capillary electrophoresis.

The interactions between eight fluoroquinolone antibiotics (ciprofloxacin, enoxacin, fleroxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, pefloxacin) and bovine serum albumin (BSA) were studied by affinity capillary electrophoresis (ACE). The binding constants were estimated by the change of migration times of the analytes through the change of concentration of BSA in the buffer solution. The yield binding constants were between 3.19 x 10(4) and 1.21 x 10(5) M(-1). These were related with the structures of fluoroquinolones, and agreed with the results obtained by other techniques. The obtained binding constants may help us in gaining some insights on possible drug/protein interactions and in early evaluation of the drugs' pharmacokinetic profiles during drug discovery.     

Contributions of hydroxyethyl groups to the DNA binding affinities of anthracene probes

Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25 degrees C) is exothermic. The binding constants increased from 1.5 x 10(4) to 1.7 x 10(6) M(-1) as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (approximately 630 cm(-1)), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (DeltaT(m) approximately 7 degrees C, DeltaDeltaH approximately 3 kcal/mol, DeltaDeltaS approximately 6-20 cal/K.mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 x 10(5) M(-1)) versus N-hydroxyethyl side chains (Kb = 5.5 x 10(5) M(-1)). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.     

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).     

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.     

孔雀石绿与牛血清白蛋白的相互作用

运用荧光光谱和紫外-可见吸收光谱研究了在缓冲溶液中不同温度下孔雀石绿(MG)与牛血清白蛋白(BSA)之间的相互作用. 实验结果表明, MG对BSA的内源荧光猝灭为静态猝灭过程. 测定了该反应在不同温度下的结合常数KA, KA分别为7.69×10⁴ L·mol-1(10 ℃)、5.31×10⁴ L·mol-1(20 ℃)和4.85×10⁴ L·mol-1(37 ℃), MG与BSA以摩尔比1:1结合. 根据Forster非辐射能量转移理论, 求出了37 ℃时给体(MG)和受体(BSA)之间能量转移效率和结合距离分别为E=0.1635 和r=2.30 nm. 计算出的热力学参数表明, MG 和BSA之间的作用力主要是通过氢键和范德华力相互作用.     

亲和毛细管电泳测定孕酮与其单克隆抗体的结合常数

采用亲和毛细管电泳的配体分离模式,以激光诱导荧光作为检测手段,测定了荧光素标记的孕酮与孕酮我隆抗体之间的结合常数,并研究了温育时间、电泳条件等因素对测定的影响。     

一种研究生物大分子与其小分子配基相互作用的毛细管电泳新方法

建立了一种定量研究大分子与小分子间相互作用的毛细管电泳新方法,即在线微透 析三电极系统亲和毛细管电泳法。本法可测定小分子的自由浓度而不受蛋白质大分子的干 扰。详细介绍了实验装置的制作,探讨了进样机制。作为实例,研究了生理条件下ｐＨ７．４, ５０ｍｍｏｌ／Ｌ磷酸介质中,牛血清白蛋白与磺胺甲基异噁唑、Ｌ－色氨酸、Ｄ－色氨酸的亲和常数的 测定,其值分别为５．８×１０~ ４Ｌ／ｍｏｌ, ２．３ ×１０~４Ｌ／ｍｏｌ, １．７７×１０~３Ｌ／ｍｏｌ。本法较简便,能在生理环 境下测定,样品用量亦少。