The kinetics of the action of pepsin on synthetic substrates.

The pepsin used in the preliminary experiments of this study was prepared from Parke, Davis pepsin by the method of Philpot (2) and was twice recrystallized. It was stored until used under half saturated magnesium sulfate solution at 2”. Pepsin A was prepared from Cudahy pepsin by the fractionation procedure of Herriott, Desreux, and Northrop (3). Its solubility in 0.05 M acetate, pH 4.6, 0.5 saturated magnesium sulfate solution was found to be constant and the same as that reported by these authors. Experiments made with this pepsin were performed immediately after it was prepared. The two alcohol-crystallized pepsins were prepared from Parke, Davis pepsin and from Cudahy pepsin by the method of Northrop (4) and were twice recrystallized. They were stored in 20 per cent alcohol at 2”. For use in the experiments, Pepsin A and the alcoholcrystallized pepsins were dissolved in 0.1 M acetate buffer, pH 4.6, and dialyzed overnight at 2” against a large volume of the same buffer. The Philpot pepsin was dissolved in 1.05 M acetate buffer, pH 4.6, and was not dialyzed. Fresh solutions of all the pepsins were prepared about every 7 days. Somewhat older solutions mere used, however, when the purpose of the experiment was purely comparative. The total nitrogen of these solutions and that not precipitated by boiling 5 per cent trichloroacetic acid were determined by the micro-Kjeldahl procedure, and the pepsin nitrogen was calculated as the difference between the two. Acetyl-L-phenylalanyl-L-tyrosine and N-acetyl-L-tyrosyl-L-tyrosine were prepared as previously described (1). That they were free from an admixture of their diastereoisomers, from which bhey had had to be separated in the course of their preparation, was shown by the fact that they were 100 per cent hydrolyzed by pepsin within a few hours, lvhereas their di-