Enzyme kinetics by Reverse Phase HPLC? - (Apr/21/2008 )
Hi Bio forumers,
I've developed an enzymatic assay in which product formation is being detected by RP-HPLC.
Does anyone know how I can obtain kinetic data (e.g. km, kcat, Vmax, etc) from my chromatograms? Do I just take the area of the peak as the rate of reaction?
Thank you in advance for any suggestions! 
need a little more detail.
if you take area of peak, how long was reaction? area/time of reaction would be rate of reaction.
if you take area of peak, how long was reaction? area/time of reaction would be rate of reaction.
I did 3 rxns at different timepoints - 3 hour, 6 hour and 24 hour incubations of enzyme + substrate. Is it possible to get kinetic information from this? If so, How? Thanks for your reply!
you may be able to get some rate info (and may be able to determine linearity of the reaction rate) if the enzyme is slow acting and the substrate concentration is saturating.
you can divide the area of the peak by the time base that you want for the evaluation (1e- if you want the change in area per hour then divide by 3, 6 and 24, respectively).
you will not be able to determine initial velocity with these time points.
To get the Km, Vmax, and kcat you will have to do the same experiment at several substrate concentrations, then plot the reaction rate vs. the substrate concentration and fit to the Michaelis-Menten equation.
Like others have said, you use the peak area and time to get the reaction rate. I would plot the three data points you have as peak area vs. time and fit to a line to get the slope (with units of area/hour). Like others have said, it's important to make sure you're getting the initial linear part of the reaction, though. I would try some earlier time points if you haven't already to make sure that's the case.
If you want the reaction rate in units of Molar/time, you can run known concentrations of product and create a standard curve (assuming you're measuring increase in the product peak and not decrease in the substrate peak, in which case you'd run known concentrations of substrate).
Hope this helps!