Usually, for fixed-time or discontinuous enzyme assay, a specific incubation period (say 30 minutes incubation period) is required before the reaction is stopped and measured (say colorimetrically). Then, how can we measure the substrate utilized or product released w.r.t time? In case of continuous reactions, the same can be monitored easily w.r.t time and hence V0 can be measure directly. But for fixed-time assays, how can the initial velocity of an enzymatic reaction be precisely calculated? Do I need to stop the reaction after specific intervals of time (by gradually increasing/decreasing incubation time)while stopping the reaction at each point of time? Thanks in advance.
Never, ever do enzyme kinetics with only one time point. Many an enzymologist career was finished because they assumed a linear velocity (product formed over time required) when in actual fact the relationship was curved: product inhibition, substrate depletion or instability of enzyme, substrate or product are only some things that can go wrong.
Ideally, you can monitor product formation or substrate depletion in real time (example: NAD(P)H dependent dehydrogenases at photometrically at 345 nm). Sometimes, it is possible to couple the reaction of interest to a reaction that can be monitored (coupled test of Warburg), for example ADP production via pyruvate kinase/lactate dehydrogenase.
If none of the above works, you need to do time points. There are two ways of doing that, either start all reactions at one time and stop them after various intervals, or start at different times and stop all at once. The former is often used when the signal is stable after stopping the reaction, e.g. in ELISA with pNPP as substrate, the reaction is stopped and the phosphatase killed with NaOH, the nitrophenol is then determined photometrically when all reactions have finished.
Thanks everyone for the clarifications. But as I have read in several peer-reviewed articles, most of them often mention a fixed time of incubation for enzyme assay before stopping the reaction and measuring absorbance. This created confusion in my mind. Please check this article which mentioned a fixed time of assay (Article Evidence for substrate assisted catalysis in N -acetylphosph...
) for measuring enzyme kinetics. Please help me to understand the assay from this article. Thanks- Badges
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