A rapid and sensitive spectrophotometric method for the assay of chymotrypsin.

For several years this laboratory has been concerned with an investigation of those enzymes in rat skin extracts which, in part at least, comprise the proteolytic enzyme system of this organ (l-5). In certain cases, methods have been proposed for the detection of their individual activities. However, certain contemplated studies, e.g. the possible physiological significance of these proteolytic enzymes in hypersensitivity and postthermal injury reactions, have indicated the need for assay procedures considerably more sensitive than hitherto used (2). Since the substrate specificities of several of the skin enzymes resemble that of chymotrypsin in that aromatic amino acid esters and their acylated derivatives, e.g. n-tyrosine ethyl ester and N-acetyl-n-tyrosine ethyl ester, serve as susceptible substrates, we attempted to devise a sensitive and rapid spectrophotometric assay procedure for these enzymes by using chymotrypsin as the test enzyme. Hartley and Kilby (6) first reported that chymotrypsin catalyzed the liberation of p-nitrophenol from the nitrophenyl ester, p-nitrophenyl acetate, despite the absence of previously established structural requirements for a chymotrypsin substrate in the acyl contributor to the sensitive bond of this compound (7). Further work by these investigators (8) and by Gutfreund and Sturtevant (9) led to the concept that a rapid mole per mole interaction of p-nitrophenyl acetate and chymotrypsin occurred with resulting formation of acetyl chymotrypsin and the concomitant release of p-nitrophenol (acylation reaction). This was followed by the much slower deacylation reaction in which active enzyme was liberated. The actual isolation of monoacetyl chymotrypsin was accomplished by incubation of the enzyme with p-nitrophenyl acetate at a pH low enough to inhibit deacylation (10, 11). Carbobenzoxyglycine p-nitrophenyl ester (12) and the nitrophenyl esters of hippuric acid, hydrocinnamic acid, and others (13) are also hydrolyzed by chymotrypsin, the efficiency of catalysis increasing as the structure of the acid moiety approaches that of a typical chymotrypsin substrate (13, 14). These observations logically led to the conclusion that a p-nitrophenyl ester of an acylated aromatic amino acid should be an extremely sensitive chymotrypsin substrate. In this paper, details for a simple, rapid, and sensitive spectrophotometric determination of chymotrypsin activity with the use of N-carbobenzoxy-n-tyrosine p-nitrophenyl ester