Binding of caffeic acid to human serum albumin by the retention data and frontal analysis

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high‐performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10⁴/m . The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm . The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.     

钳蝎毒中抗癫痫肽、镇痛肽和抗肿瘤肽的快速同时分离和鉴定

采用Shim-Pack WCX-1型阳离子交换高压色谱柱对中国东亚钳蝎全蝎毒进行了分离，在鉴定了其中的抗癫痫肽、镇痛肽和抗肿瘤肽活性峰的基础上，应用Shim-Pack DIOL-300型凝胶排阻高压色谱柱对它们进行了进一步分离和鉴定，可以得到较纯的3种多肽。在高压色谱所提供的全蝎毒分离信息的基础上，应用与Shim-Pack WCX-1色谱柱具有相同交换基团的、具有较大吸附容量的CM Sepharose CL-6B软胶介质在低压色谱上对全蝎毒进行了分离，并分别对其中的抗癫痫肽、镇痛肽和抗肿瘤肽进行了鉴定。     

用前沿色谱法测定蛋白质吸附等温线时增浓方式的研究

用连续前沿色谱法和断续前沿色谱法测定了七种标准蛋白在疏水色谱填料上的吸附等温线,两种方法之间存在着一定的差别。从流出曲线突跃处斜率测定的不确定性、色谱过程中动力学因素和实验方法本身存在的问题等几个方面探讨了误差的来源,指出了可能解决这一问题的途径。     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

两步柱色谱法分离纯化重组肿瘤血管生长抑制因子Kringle 5

建立了一种用Ni2+螯合的Chelating Sepharose Fast Flow亲和柱色谱和Sephadex G-75凝胶排阻柱色谱分离纯化重组肿瘤血管生长抑制因子Kringle 5的方法。采用该工艺得到的重组Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明其纯度约为98%,且具有抑制鸡胚绒毛尿囊膜新生血管生成的生物活性。     

Investigation on the Binding of Terazosin Hydrochloride and Naftopidil to an Immobilized α 1-Adrenoceptor by Zonal Elution

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α ₁-Adrenoceptor (α ₁-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α ₁-AR with an association constant of 1.45 × 10⁶ M^?1 and a concentration of binding sites of 1.56 × 10^?6 M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 10⁵ M^?1 and 2.06 × 10³ M^?1, and the corresponding concentrations of the binding sites are 1.56 × 10^?6 M and 1.11 × 10^?3 M, respectively. It is concluded that the stationary phase containing attached α ₁-AR can be used to realize the binding of a drug to the receptor.     

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature‐induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed‐phase liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12–50 °C. The results showed that, in the temperature range 12–20 °C, BSA only existed in a single conformation state A, while in the temperature range 22–50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy‐driven. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Investigation on the Binding of Terazosin Hydrochloride and Naftopidil to an Immobilized α 1-Adrenoceptor by Zonal Elution

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α ₁-Adrenoceptor (α ₁-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α ₁-AR with an association constant of 1.45 × 10⁶ M⁻¹ and a concentration of binding sites of 1.56 × 10⁻⁶ M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 10⁵ M⁻¹ and 2.06 × 10³ M⁻¹, and the corresponding concentrations of the binding sites are 1.56 × 10⁻⁶ M and 1.11 × 10⁻³ M, respectively. It is concluded that the stationary phase containing attached α ₁-AR can be used to realize the binding of a drug to the receptor.

     

Urea-Induced Dissociation of Nerve Growth Factor from Venom of Chinese Cobra by SEC

The urea-induced dissociation of nerve growth factor from venom of Chinese cobra (_cNGF) was studied by intrinsic fluorescence emission spectra, SEC, urea-gradient polyacrylamide gel electrophoresis, assays of biological activity and thermodynamic parameters. The results showed that when urea concentration was lower than or equal to 4.0 mol L⁻¹ or higher than or equal to 8.0 mol L⁻¹, _cNGF existed only in native homodimer form or monomer form, respectively; whereas when urea concentration was higher than 4.0 mol L⁻¹ and lower than 8.0 mol L⁻¹, they existed simultaneously in the native homodimer and monomer forms and the former decreased, while the latter increased with the increase in urea concentration. Based on the association–dissociation equilibrium between _cNGF and urea molecules, an equation, which includes two characteristic dissociation parameters K and ∆m, was presented to describe the urea-induced dissociation process of _cNGF. As the reaction temperature increased from 15 to 35 °C, positive enthalpy and entropy changes were observed, and the parameter K increased from 2.72 × 10⁻¹³ to 5.18 × 10⁻¹² (L mol⁻¹), while the parameters ∆m and ∆G, respectively, decreased from 10.18 to 8.42 and from −10.27 to −18.67 (kJ mol⁻¹), which means that the urea-induced dissociation of _cNGF was spontaneous and entropy-driven and the higher temperature was favorable for the dissociation process. Using the procedures and equations mentioned in the paper, the urea-induced dissociation of _cNGF is first comprehensively described. Furthermore, this work presents a useful method for people to study the dissociation of dimer or multimer proteins induced by denaturants, inducers, pH, etc.