Activities of Directly and Indirectly Acetylated Chymotrypsins.

The introduction of one acetyl group into the chymotrypsin molecule by reaction with p-nitrophenyl acetate gives rise to an enzymatically inactive monoacetylated protein that rapidly loses its acetyl group and becomes active again in solutions of dilute alkali or primary alcohols." 2 On the other hand, the well-known polyacetylation of proteins with acetic anhydride, as described by Fraenkel-Conrat, Bean, and Lineweaver,' yields active (or potentially active) products with chymotrypsin and chymotrypsinogen in which much of the acetyl remains rather firmly bound. The following paper is meant to give a first account of some observations made on polyacetylated chymotrypsinla and chymotrypsinogen that may be of interest because they indicate that these artifacts behave enzymatically on several counts in very different fashion from their naturally occurring counterparts. Marked differences between the behavior of derived chymotrypsins and the naturally occurring enzyme were observed by Jansen et al.4 who found the pH optimum of esterolysis4a to be altered by acetylation. They also observed that esterolytic activity is much less reduced by oxidation with periodate than is proteolytic activity. The chemical modifications made by Vallee et al.' on carboxypeptidase A have led to even more striking changes in the properties of that enzyme. The peptolytic activity may be caused to disappear altogether, while at the same time the esterolytic activity increases manyfold. Multiple acetylation of chymotrypsin led in our hands to a product of mediocre activity, compared to the original enzyme, on both TEE and protein. However, when a solution was maintained at neutrality for some time, its esterase activity approximately doubled and thus became about equal to that of the natural enzyme. The proteolytic activity remained unchanged. Observations on chemically modified chymotrypsinogens, including acetylated chymotrypsinogen, have been reported by Wilcox in a series of papers.'9 They leave no doubt that in many instances such modified proteins yield active estersplitting enzymes on treatment with trypsin. In the case of the acetylated zymogen, the observed esterolytic activity was as high as that obtained from natural chymotrypsinogen under like conditions.7 Since some of the groups in active chymotrypsin, including the so-called "active center" itself, are evidently not available for reaction in chymotrypsinogen, we thought it might be of interest to compare the active protein obtained by the direct acetylation of chymotrypsin with that obtained by activation of the previously acetylated zymogen. It was found that acetylated chymotrypsinogen differed from the naturally occurring protein in its behavior with trypsin. Unlike the natural protein, it formed little or no active enzyme with trypsin at pH 7.0-7.2, unless an electrolyte was also present in appreciable concentration or, alternatively, some natural (i.e., not acetylated) chymotrypsin. MXloreover, the product of activation differed from both natural and directly acetylated chymotrypsins in that its estero-