Have anybody measured a kinetics of enzymatic digestion of BAEE(n-alpha-Benzoyl-Arginine Ethyl Ether) with USP method? I have observed acceptable delta-OD when use significant amounts of enzyme but now I have a problem with measuring kinetics of blank and sample of protein with no Arg-C activity. It is quite difficult to get absolute straight line and it seems to be non-enzymatic hydrolysis of substrate with very low velocity (especially in comparison with trypsin and clostripain). Should I further work on getting near zero delta with blank and what can be the solvation of the problem?
BAEE is an ester (not an ether). Some level of spontaneous hydrolysis is to be expected, especially if the pH of the reaction is either acidic or basic. All you have to do is subtract the rate of spontaneous hydrolysis from the rate of enzyme-catalyzed hydrolysis to remove the spontaneous background rate.
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