Studies on the reduction and re-formation of protein disulfide bonds.

Previous communications have reported on the reduction of the disulfide bonds of ribonuclease (14)) ribonuclease-S-protein (5), and lysozyme (6) by treatment of the native protein or protein derivative in 8 M urea solutions with thioglycolic acid or mercaptoethanol. Reduction by this procedure has a significant advantage over certain other techniques’ (e.g. borohydride (7) or sulfite reduction (8)) in that it yields reduced molecules that have suffered minimal, if any, covalent change other than that involved in the cleavage of the disulfide bonds to sulfhydryl groups. Thus, it has been observed’ that fully reduced ribonuclease contains no new NH&erminal amino acids, and that its molecular weight and amino acid content (after alkylation of the SH groups) are in accord with the expected values. It is therefore possible to study the re-formation of disulfide bonds and to examine the influence of chemical and environmental modifications on the efficiency of reoxidation and reactivation. It has been demonstrated that the eight sulfhydryl groups of fully reduced ribonuclease and of ribonuclease-S-protein undergo spontaneous oxidation to yield macromolecules that are extremely similar, and probably identical, to the parent molecule in their physical and enzymatic properties (4, 5). The methods of reduction and subsequent manipulation that we have reported have, over the past year, been somewhat modified. We would like to summarize here some of the aspects of reduction, sulfhydryl group stabilization, and alkylation that appear to be of general use in the reduction of proteins, m well as some observations on conditions for regeneration of diiulfide bonds in reduced ribonuclease.