Interaction of Phosphorothioate with the Disulfide Bonds of Ribonuclease and Lysozyme.

Disulfide bonds are undoubtedly of great importance in stabilizing the native conformations of many proteins. For studies of the covalent structure of proteins it is desirable, as a first step, to cleave the disulfide bonds (1). This has been carried out either by oxidation (l-3) or by reduction followed by alkylation of the sulfhydryl groups formed (4). Iodoacetic acid (or iodoacetamide) has been widely used for the alkylation step, and has the advantage that in its radioactive form it can serve as a marker for the sulfhydryl groups with which it reacts. The reductive cleavage of disulfide bridges in biologically active proteins may result in the loss of their activities. These activities may, in some cases, be recovered upon the spontaneous reosidation of the reduced proteins to their native forms (5-18). In studies on the correlation of structure with biological function, it is disadvantageous to block the sulfhydryl groups in an irreversible manner. Looking for a reagent that would quantitatively cleave disulfide bonds, react with and stabilize protein sulfhydryl groups, be obtained easily with a radioactive label, and be removable from the protein under relatively mild conditions, we have tested trisodium phosphorothioate (monothiophosphate) (19, 20). This reagent has recently been shown to act as a reducing agent, to cleave the disulfide bonds of cystine and glutathione; and to activate the enzymes papain and ficin.2 In the study presented here, it is shown that the disulfide bonds of bovine pancreatic ribonuclease and egg white lysozyme are cleaved by phosphorothioate. The stoichiometry of the reaction, the stability of the phosphorothiolated enzymes, and the removal of phosphorothio groups are also described.