The relationship of disulfide bonds and activity in ribonuclease.

The RNase molecule consists of a single chain, arranged in a compact, folded structure, cross-linked through 4 disulfide bridges (1). After hydrolysis to determine the amino acid composition of RNase, 8 half-cystine or cysteic acid residues have been identified (2,3) and the approximate location of these residues in the partial structural formula for oxidized RNase has been determined (4). More recently, the elucidation of the pairings of these 8 cysteic acid residues has been accomplished. Numbered from the N-terminal end of the protein chain, the presence of 4 disulfide bridges has been established, linking halfcystines 1 and 6, 2 and 8, 3 and 7, and 4 and 5 (5, 6). Total cleavage of the 4 disulfide bridges in RNase, whether by oxidation with performic acid (3) or by reduction with thioglycolic acid (7), leads to complete loss of enzymatic activity (3, 7). Activity still remains high with cleavage of 1 or 2 disulfide bonds in the molecule but decreases rapidly with reduction of more than 2 disulfidc bonds (7). In an extension of previous studies relating secondary structure to activity for the enzyme RNase (8), we attempted to evaluate the influence of the -S-Sbonds on the secondary structure and activity of the enzyme. The amperometric technique for sulfhydryl determinations has been adapted, with modifications, to the measurement of disulfide bonds in proteins (9). With this procedure, 1 of 4 disulfide bridges in RNase was found to be resistant to cleavage. Further modifications such as carrying out the sulfitolysis reaction and titration in various concentrations of urea up to 8 M, have resulted in a relatively simple, rapid and accurate method for the controlled cleavage of all the disulfide bonds in many proteins, including RNase (10). By this method, we have attempted to correlate enzyme activity with the integrity of the disulfide bonds present in RNase. This communication presents evidence that not all of the disulfrde bridges are essential for activity of RKase, confirming the results of Sela et 01. (7) obtained with a different method. In addition, evidence is presented which strongly suggests that it is not only the cleavage of disulfide bonds, but in addition, a subsequent structural alteration in the molecule, in the denaturation sense, which is responsible for loss in enzymatic activity.