I'm performing a chromogenic substrate assay to analyze a trypsin inhibitor. I'm wondering whether it's necessary to also perform an active site titration of the trypsin itself (bought from Sigma). I've been told it's not necessary, but have read papers asserting the contrary. What is considered the best practice?
If the inhibitor is very potent, so that you see significant inhibition at concentrations near the enzyme concentration, then you will need to know the active site concentration in order to calculate the inhibitor's potency correctly. But if the inhibitor is not so potent, it is not that important to know the active site concentration to high precision.
From my point of view active site titration is crucial and very simple procedure before you start to work with trypsin. The active form's amount could differ from preparation to preparation so if the experiments is time or trypsin consuming you should titrate it. Good luck
Similar to an answer above, active site titrations, at least for getting a simple Km, Ki and Vmax values are not needed unless you have a very strong binding inhibitor and also want to do advanced kinetic calculations.
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