The action of chymotrypsin on two new chromogenic substrates

Abstract

Two new chromogenic substrates of chymotrypsin have been synthesized: N -succinyl- L -phenylalanine p -nitroanilide (SPNA) and N -glutaryl- L -phenylalanine p -nitroanilide (GPNA). Both are stable in the absence of enzyme and release p -nitroaniline (yellow) upon hydrolysis. GPNA is the more sensitive substrate being capable of quantifying less than 10 μg per milliliter chymotrypsin. The presence of Ca ++ decreases K m of the enzyme-substrate reaction but has a negligible effect upon k 3 . Na ++ , on the other hand, increases k 3 as well, and provides a suitable means of increasing the sensitivity of GPNA down to microgram levels of enzyme. In combination with an “all or none” assay the rate of hydrolysis of GPNA can be related to the molar concentration of enzyme. The reaction of chymotrypsin with GPNA is not subject to substrate activation over a 200-fold range of substrate concentration, nor is it affected by the presence of ethyl ammonium chloride. In these respects it differs from the reaction of trypsin with susceptible substrates.