【作者】 熊彩侨；

【导师】 夏之宁；

【作者基本信息】 重庆大学 ， 分析化学， 2009， 硕士

【摘要】 动力学毛细管电泳（kinetic capillary electrophoresis, KCE）是新发展起来的用于亲和相互作用研究的新方法,相对于传统的亲和方法,KCE集中了均相分析方法的简单性以及动力学分析方法的动力学特征,可以作为亲和测定和亲和纯化的均相动力学方法的概念平台。但是传统KCE在计算方法及实验操作中都存在一些不足。本文针对传统KCE的这些不足,发展了几种更简单的KCE新方法,并将KCE的应用拓展到了蛋白及DNA与药物的相互作用研究中。本文的主要研究内容为:第一部分:测定动力学参数的平衡混合物的非平衡毛细管电泳（NECEEM）和区段-区段动力学毛细管电泳（ppKCE）新方法的建立及应用。在传统KCE的基础上,从动力学参数k_on、k_off的原理出发,利用物质的峰高表示其浓度,建立了NECEEM及ppKCE测定动力学参数的新方法,并将这些方法应用到了西酞普兰与BSA的相互作用研究中。利用NECEEM测得了离解速率常数k_off,并通过考察结合物离解时间与浓度参数ln（1/h_c）之间的线性关系以及实验方法的精密度、稳定性和重现性,验证了k_off求解方法的可靠性。采用建立的ppKCE新方法同时测得了动力学参数k_on和k_off以及结合常数K,测定的结果通过时间比法得到了验证。第二部分:校正的预平衡区带毛细管电泳（modified pre-eq CZE）新方法的建立及应用。传统的pre-eq CZE由于电泳中结合物的离解而存在对结合常数K值测定偏小的缺陷,因此文中将NECEEM与pre-eq CZE相结合,根据pre-eq CZE中结合物的离解过程,首先对离解速率常数k_off进行了求解,进而利用所求得的k_off对药物游离浓度的测定进行校正,建立了modified pre-eq CZE新方法,并将其应用到了西酞普兰与BSA以及硝基苯胺与鲱鱼精DNA间的相互作用研究中,同时将利用modified pre-eq CZE求得的结合常数与毛细管电泳前沿分析法、峰漂移法、紫外-可见吸收光谱法及平衡透析法所得的结果进行了对比,从而对新方法中校正的正确性和可靠性进行了验证。这种新方法能够消除传统pre-eq CZE仅适合于低离解速率相互作用研究的缺陷,并能够同时测得动力学参数及结合常数。本文所建立的测定动力学参数及结合常数的动力学毛细管电泳方法简单、可靠,为药物与生物大分子的亲和相互作用研究提供了新方法。更多还原

【Abstract】 Kinetic capillary electrophoresis （KCE） is a novel method for the study of affinity interaction. Comparing to the traditional affinity method, KCE combines the simplicity of homogeneous methods with enabling kinetic features of kinetic methods and can be regarded as a conceptual platform for the affinity determination and purification methods. However, there are some limitations in both the calculation method and experimental operation of traditional KCE. Considering these shortages of traditional KCE, several simpler new methods of KCE were established in this paper, and the application of KCE was expanded to study the interaction between drugs and protein or DNA. The main content of the paper is summarized as follows:First part: Establishment and application of non-equilibrium capillary electrophoresis of equilibrium mixture （NECEEM） and plug-plug kinetic capillary electrophoresis （ppKCE） new methods for the determination of kinetic parameters. Based on the principle of kinetic parameters k_on, k_off and let the peak heights represent its concentrations, the new NECEEM and ppKCE methods were developed for the determination of kinetic parameters. Then both the new methods were used to study the interaction between citalopram and BSA. In NECEEM, the value of k_off was obtained and the reliability of this method was confirmed by the linear relationship between the dissociation time of complex and the concentration parameter ln（1/hc）, and by the investigation of precision, stability and repeatability of methodology. In the established ppKCE, the kinetic parameters k_on, k_off and the binding constant K were simultaneously determined, and the results were verified by time ratio method.Second part: Establishment and application of modified pre-equilibrium capillary zone electrophoresis （modified pre-eq CZE）. In traditional pre-eq CZE, smaller binding constant K will be determined because the complex dissociated in the electrophoresis. So the modified pre-eq CZE method was established in this paper by combining NECEEM and traditional pre-eq CZE. In the new method, the dissociation rate constant k_off was firstly determined according to the dissociation process of complex and then used to modify the determination of the concentration of unbound drug. The interactions between citalopram and BSA, nitroaniline and herring sperm DNA were studied by this new method. In addition, the accuracy and reliability of the method were confirmed by comparing the value of binding constant determined by modified pre-eq CZE with that obtained by capillary electrophoresis frontal analysis, mobility shift analysis, uv-vis spectrophotometry and balance percolation method. This new method not noly can eliminate the disadvantage of traditional pre-eq CZE that it only adapted to the interaction system with slow dissociation kinetics, but can determine the kinetic parameters and binding constant simultaneously.The novel kinetic capillary electrophoresis methods for the determination of kinetic parameters and binding constant were established in this paper. They are simple and reliable, and provide new methods for the study of the affinity interaction between drugs and biomacromolecule.更多还原