Crystalline Pepsin Iii. Preparation of Active Crystalline Pepsin from Inactive Denatured Pepsin By

The results (1) of the experiments with crystalline pepsin isolated from crude pepsin preparations indicated that the material is a pure substance and that the proteolytic activity is a property of the protein molecule itself and is not due to the presence of a separate non-protein impurity. No indication of the presence of a more highly active nonprotein molecule was obtained in the solubility measurements, inactivation experiments, or measurements of the rate of diffusion. In a sense however, this evidence is all negative in that it merely fails to show the presence of a more active molecule. I t was shown, for instance, that the loss in activity in alkaline solution was quantitatively paralleled by the production of insoluble denatured protein. I t could be objected, however, that the enzyme was liberated from the protein when the latter became denatured and that the enzyme itself was too unstable to exist alone. If it could be shown, however, that reversal of denaturation of the protein was accompanied by reactivation of the enzyme and that the native protein obtained in this way had the same activity as the original protein, strong proof would be furnished for the assumption that the activity was really a property of the protein molecule. In order to account for this result on the hypothesis that the activity was due to the presence of a small amount of a more highly active substance it would be necessary to suppose that the conditions for transforming the denatured protein into the original pro713