Establishment Of Novel Kinetic Capillary Electrophoresis And Its Application In The Study Of Interaction

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.