Acetyl chymotrypsin and its reaction with ethanol.

A short report has recently been made (1) concerning the preparation of an acetyl derivative of chymotrypsin containing a single acetyl group of relatively high reactivity. The reaction, which may be carried out by any of several nitrophenyl acetates, begins with a rapid initial liberation of the free nitrophenol in practically mole for mole proportions to the total chymotrypsin present. Thereafter a slow but continuous breakdown of the acetate takes place at a speed approximately proportional for some time to the concentration of enzyme present. This behavior of chymotrypsin was first noted by Hartley and Kilby (2), who concluded from their study that a monoacetylated chymotrypsin was rapidly formed and less rapidly decomposed. Chymotrypsin is known to be unusually resistant to acid, and in repeating this reaction we observed that the initial “burst” of free nitrophenol still occurred at pH levels at which the subsequent breakdown was so slow as to be nearly negligible. This observation naturally suggested that the intermediate could perhaps be isolated, and subsequent trial proved that this was indeed the case. In their reaction with chymotrypsin, the nitrophenyl acetates resemble phosphorylating agents such as diisopropyl fluorophosphate and analogous substances (3) in that the reaction is mole for mole, leads to an inhibited end-product, and does not occur, under comparable conditions, with chymotrypsinogen, denatured chymotrypsin, or phosphoryl chymotrypsin. The acetylated enzyme differs from the phosphorylated enzyme, however, in being easily decomposed, for example by dialysis against water. Decomposition in dilute alkali (pH 7 to 8) is almost immediate, and the activity of the previously inhibited enzyme is recovered quantitatively. The present paper reports some of the methods employed in this work as modified by recent experience. Moreover, it has been observed that acetyl chymotrypsin is able to acetylate OH groups, and the acetylation of ethanol has been especially studied.