Crystalline Acetyl Derivatives of Pepsin

Crystalline pepsin has been acetylated by the action of ketene in aqueous solution at pH 4.07-5.5. As acetylation proceeds the activity decreases, the decrease being more rapid at pH 5.0-5.5 than at 4.0-4.5. Three acetyl derivatives have been isolated from the reaction mixture and obtained in crystalline form. The crystal form of these derivatives is similar to that of pepsin. Fractionation and solubility determinations show that these preparations are not mixtures or solid solutions of the original pepsin with an inactive derivative. A compound which contains three or four acetyl groups and which has lost all of its original primary amino groups can be isolated after short acetylation. It has the same activity as the original pepsin. A second derivative containing six to eleven acetyl groups has also been isolated. It has about 60 per cent of the activity of the original pepsin. A third derivative having twenty to thirty acetyl groups and about 10 per cent of the activity of original pepsin can be isolated after prolonged acetylation. The 60 per cent active derivative on standing in strong acid solution loses some of its acetyl groups and at the same time regains the activity of the original pepsin. The compound obtained in this way is probably the same as the completely active three acetyl derivative obtained by mild acetylation. These results show that acetylation of three or four of the primary amino groups of pepsin causes no change in the specific activity of the enzyme but that the introduction of acetyl groups in other parts of the molecule results in a marked loss in activity. The solubilities, amino nitrogen content, acetyl content, isoelectric point, and the specific activity have been determined by a variety of methods and found to be different from the corresponding properties of crystalline pepsin. The pH-activity curves, acid and alkali inactivation, and titration curves were not significantly different from the same respective properties of pepsin.