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

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(i) 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.