Studies on the Enzymatic Breakdown of Proteins II. THE ACTION OF PEPSIN ON RIBONUCLEASE AND CONSIDERATIONS ON THE STRUCTURE OF RIBONUCLEASE* t

The susceptibility of ribonuclease to pepsin was demonstrated some time ago by Kunitz (1) and interesting conclusions about the structural basis of RNase activity have been derived from subsequent investigations by Anfinsen (2, 3). Since RNase is a single polypeptide chain (4) of 124 amino acids (5) held in a compact configuration largely through the restraint imposed by four disulfide bonds (4, 6, 7), it seemed that this protein would serve as an interesting model substrate for studies of the enzymatic breakdown of macromolecules. Moreover such studies should reveal information about the relative roles of the primary, secondary and tertiary structures in a protein. In a previous paper (8) it was shown that the chymotryptic digestion of insulin proceeds by the one-by-one mechanism (9). No molecules of a size intermediate between the starting material and the polypeptides produced by exhaustive enzymatic treatment could be detected in the partial digest. In contrast, during the digestion of RNase by pepsin, intermediates were detected readily, and some of the properties of these molecules have been studied. It was found, for example, that partially digested RNase is still active in hydrolyzing ribonucleie acid and that the enzymic activity resides in molecules substantially smaller than the intact molecules. Further degradation causes a loss in activity leading ultimately to polypeptide fragments with a molecular weight about 4,060 (as compared to 13,683 (5) for native RNase). During the early stages of the degradation of RNase some of the disulfide bonds become more susceptible to reduction than are the relatively inaccessible disulfide bonds of native RNase. The digestion appears to be an example of the zipper mechanism described by Linderstrom-Lang (10).