A bypass trapped-flow analysis system evaluation of enzyme kinetic parameters with a coupled enzyme assay and fluorescence detection

摘要

The newly developed flow instrument, bypass trapped-flow analysis system (ByT-FAS), was used to perform enzyme assays and generate data for evaluation of an enzyme's kinetic parameters. The enzyme assay was yeast hexokinase coupled to glucose-6-phosphate dehydrogenase and the production of reduced NADPH was monitored fluorometrically. The kinetic parameters of hexokinase that were evaluated with the system were the apparent K_m's of the substrates glucose and ATP, and the K, of the competitive inhibitor for ATP, ADP. The ByT-FAS generated data were comparable to other manually derived published values of the kinetic constants for hexokinase and validates the utility of the ByT-FAS system for making absolute enzyme measurements. The semi-automated ByT-FAS system can perform analyses more rapidly than manual assay techniques with less manual manipulation and smaller sample and/or reagent volumes.