The oxidation of ribonuclease with performic acid.

In studies on the chemical structure of ribonuclease in which trypsin is used as a specific reagent for hydrolysis (2), it has been found necessary first to cleave the disulfide bridges in the molecule by oxidation with performic acid in a manner similar to that employed by Sanger (3) in his investigation of insulin. Oxidation is required in order to render the protein more readily susceptible to the action of trypsin, which attacks the unoxidized molecule only very slowly.’ In contrast to the situation with insulin, cleavage of the -S-Sbridges in ribonuclease does not liberate peptide chains, since the studies of Anflnsen and his associates (4)) together with quantitative amino acid analyses carried out in this laboratory (5), indicate that the molecule is composed of a single chain of 126 amino acid residues. The cleavage of the four -S-Sbonds prior to enzymatic hydrolysis is likely to result in the production of simpler peptides than would be formed were the cross-links provided by the disulfide bridges left intact. From the data of Toennies and Homiller (6), performic acid might be expected to react to some extent with many amino acids in addition to its predominant action upon cystine, tryptophan, and methionine. The present investigation was undertaken to determine quantitatively the stability of each of the amino acid residues in the ribonuclease molecule under the conditions employed for the oxidation of the cystine sulfur bridges. For this purpose, complete amino acid analyses have been performed on hydrolysates of oxidized ribonuclease and the results have been compared with those obtained on the native protein (5). The study has been greatly facilitated bythe fact that ribonuclease, like insulin, contains no tryptophan. The presence of methionine (5), however, which doea not occur in insulin, has made it necessary to establish that all 4 methionine residues are transformed quantitatively to methionine sulfone under the conditions used in the present experiments.