A sensitive high-performance liquid chromatographic assay for trypsin-like enzymes

There is an increasing interest in trypsin-like proteolytic enzymes of physiological significance. Indeed, although present in very small quantities, these enzymes play important roles in several biological reactions such as digestion, blood clotting, fibrinolysis, complement action, fertilization and kinin formation. Thus very sensitive microassays are necessary for monitoring and studying the processes in which these enzymes are involved. N alpha -benzoyl-L-arginine ethyl ester (BAEE) is one of the most widely used synthetic substrates. The rate of hydrolysis can be determined by different methods. The consumption of alkali required to neutralize the free carboxylic groups of N alpha -benzoyl-L-arginine (BA), the product of hydrolysis, can be measured. The present communication describes the measurement of the released BA at 235 nm by directly injecting the incubation medium on to a HPLC column. The separation achieved on the reversed-phase system avoids interference by the unhydrolysed substrate without long extraction procedures or chromogenic reactions.