I am doing enzymatic reactions recently and then check the amount of product in the end by HPLC/MS. The wired thing is that reactions with shorter reaction time show higher amount of product. It is a very old enzyme that literatures show no reverse reaction by this enzyme. could someone give me some hits that why this happened? Thanks a lot !!!
A reaction has occurred that has generated an enzyme inhibitor whose concentration increases with prolonged incubation and target enzyme activity reduced resulting in less product formation from its catalysis.
Are you working with a purified enzyme or with some crude extract ? In the latter case, it may be that the product that you are assaying is converted into some other compound by another enzyme present in the system. Alternatively, do you know that the product formed by the reaction is stable ?
It could be that the reaction product is not stable over time. Doing a time course where you take samples of the reaction at different time points and run each one on HPLC/MS could be informative.
Never, ever, do an enzymatic assay by endpoint. Always do a time-course, determine that it is linear and then use the slope as reaction rate. There are too many things that can go wrong: the enzyme, the substrate or the product may be unstable, there may be product inhibition, side-reactions, ...
Now, if I take the info you provided as indication that the time-course is non-linear, and if I assume further that you have excluded substrate depletion (sometimes 10% can be enough to cause non-linearity) as obvious cause, then there are three things you can do:
Stability of the product and the purity of enzyme solution or extract may be responsible for decreasing the product concentration.
Monitor the reaction during the initial period of reaction where activity shows a linear response with increase in the time of incubation of enzymatic
reaction.
With increase in time the concentration of product increases and it might be inhibiting the enzyme activity
Thanks for everyone. I think it is very important to find the linear range. Now my result gets better.
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