High Temporal Resolution Monitoring Of Enzyme Reaction And Inhibition Using Optically Gated Vacancy Capillary Electrophoresis And Immobilized Enzyme

Since Banke et al. first used it for monitoring alkaline protease activity, capillaryelectrophoresis (CE) has become a powerful approach for quantitative studies ofenzymatic reactions. Due to its several unique advantages, such as high efficiency andsensitivity, fast analysis, extremely low sample volume requirement, and ability toutilize several detection methods, CE has been widely applied in almost all aspects ofenzyme assays, including the evaluation of the enzymatic activity, enzyme kinetics,enzyme inhibition and activation, and the investigation of enzyme-mediated metabolicpathways, etc. In the study, we present a novel method for monitoring of enzymereaction and inhibition with high temporal resolution by using optically gated vacancycapillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detectionand immobilized enzyme. Trypsin cleavage reaction and inhibition were investigatedby the presented OGVCE-LIF assay. It includes some parts in this research work：(1) Establishing the experiment device, reasonable combine the OGVCE and LIFdevice.(2) The OGVCE-LIF method was first used to analyze FAM-labeled angiotensin orFAM-Asp-Arg-OH to test the performance of the method and to obtain the calibrationcurve for quantitative measurement of the enzymatic reaction.(3) Use OGVCE-LIF method automatically monitor the enzymatic reaction with hightemporal resolution to obtain the rate constant and Michaelis constants of the trypsincleavage reaction.(4) Research the inhibition reaction of immobilized trypsin with the OGVCE-LIFassay, using two different inhibitors。...