High Performance Capillary Electrophoresis In The Dihydrofolate Reductase Reaction Kinetics

Capillary electrophoresis is a convenient tool for studying kinetics of enzymatic system due to many attractive features such as low sample volume, easy to operate, high separation efficiency and resolution, minimal reagent use, and short analysis times. In this paper, the application of capillary electrophoresis method in the research of kinetics of enzyme reactions was introduced, including three parts in details: the measurement of kinetic parameters of enzyme reactions, the research of kinetics of inhibitory activities and the separation anddetection of dihydrofolate reductase.MEKC has been applied for the study of the separation of dihydrofolate, tetrahydrofolate, NADP, NADPH and dihydrofolate reductase in the reaction system. The five components were separated in 18min by 50mmol/L disodium tetraborate (pH9.18) buffer solution with 0.002% Brij-35.A method was developed to determine dihydrofolate reductase activity by monitoring its product, tetrahydrofolate. The aim of this paper is to establish an analysis model for determination of dihydrofolate reductase by High Performance Capillary Electrophoresis.A new assay of dihydrofolate reductase using capillary electrophoresis (CE) has been established. The five components were separated in above conditions. A method was developed to determine kinetics parameters of dihydrofolate reductase by monitoring the changes of the concentrations between dihydrofolate and tetrahydrofolate. By determining the activity of dihydrofolate reductase inhibitors (DHFRI), it was found that the IC50 of aminopterin and other inhibitors matched the values had been published, and this method could be used for the screening of anticancer drugs.A method of separation the dihydrofolate reductase by capillary electrophoresis was established. Monitoring every extract process by 50 mmol/L disodium tetraborate (pH9.18) buffer solution with 0.05% carboxymethyl cellulose, the separation temperature is 25℃, the pressure is 25KV, and the detection wave is 214nm. To serve as evidence, the extract of dihydrofolate reductase is a purified process.In addition, investigates and compares systematically characteristics and applications of determination of trace folic acid by three fluorometric systems., including of the intrinsic fluorescence of folic acid, KMnO₄—irradiation system and H₂O₂—irradiation system. With the comparation, It was found that the intrinsic fluorescence of folic acid was too weak. However, the results of derivative systems were satisfied, which could improve the sensitivity to 2³...