hexokinase assay - (Aug/02/2006 )

hi,guys.
I really really need hexokinase assay. anybody want to share?????

MartyLab, here's an idea. Hexokinase catalyzes the formation of Glc-6-Phospahate from Glc + ATP. Difficult to measure anything there. So you couple that reaction with a reaction that you can measure: G-6-P + NADP (with G-6-P dehydrogenase) to form NADPH and 6-phos-gluconate. All in one test tube. Set up a standard curve using known hexokinase concentrations. Probably you'd want to check the linearity.

The INCREASE in NADPH is easily measured at 340nm spectroscopically. The hard part is working out what concentrations of the substrates to use to put them in excess and leave hexokinase as the rate limiter.