A colorimetric micromethod for the estimation of chymotrypsin activity.

The structural specificity requirements of chymotrypsin have been well defined and reviewed by several investigators (l-3). As in the case of carboxypeptidase (4), the specific amino acid required in a substrate for chymotrypsin is tyrosine, tryptophan, or phenylalanine, in order of decreasing susceptibility of their corresponding ester or peptide derivatives. The enzyme is reported to attack, in order of decreasing activity, either ester, hydroxamide, glycinamide, amide, hydrazide, or glycylglycylamide bonds involving the carboxyl group of the specific amino acid (3, 5). The presence of a free a-amino group in the vicinity of the susceptible bond is partially inhibitory to attack by chymotrypsin, and hence simple dipeptides are not the most suitable substrates for the detection and estimation of the activity of this enzyme. On the other hand, if both a-amino hydrogen atoms are replaced, as in the phthalimide derivative, all susceptibility to enzymatic hydrolysis is lost (6). It has generally been found that replacement of 1 hydrogen of the a-amino group with benzoyl, nicotinyl, acetyl, carbobenzoxyglycyl, or glycyl substituents leads, in order of decreasing activity, to greater susceptibility to oc-chymotrypsin (3). On the basis of the above information regarding the substrate specificity requirements of chymotrypsin, a series of synthetic compounds fulfilling these conditions, and at the same time incorporating a chromogenic group (/?-naphthylamine or P-naphthol) in such a manner that it would be released upon enzymatic hydrolysis of the parent compound, was prepared and characterized earlier (7). 2 moles of naphthol are coupled with 1 mole of tetrazotized diorthoanisidine, the resulting azo dye is extracted into ethyl acetate, and the color density is measured in a photoelectric colorimeter (Klett). The azo dye principle of this method has been applied to the measurement of a number of other enzymes (4,8-12). It is the purpose of this paper to describe studies on the synthetic substrates previously