A Study of the Factors Influencing the Rate and Extent of Enzymic Reactivation during Reoxidation of Reduced Ribonuclease

The rate and extent of return of enzymic activity during oxidation of the sulfhydryl groups of reduced ribonuclease were studied under a variety of conditions. The rate of reactivation was found to be inversely proportional to the concentration of reduced protein. Under the most favorable conditions employed in this study (0.01 mg. protein/ ml., pH 8.2, 24” C.), enzymic activity was demonstrable within 5 min. and full restoration of activity was attained within 60 min. Studies of the sedimentation behavior of r ibonuclease during the course of reoxidation indicated that intermolecular disulfide bond formation occurs early in the reoxidation process. In agreement with the hypothesis that the native configuration is the thermodynamically most stable one, the aggregated material subsequently undergoes a spontaneous conversion to the fully active monomer. These findings indicate that the marked delay in reactivation previously observed with more concentrated solutions of the enzyme is not an inherent feature of the reoxidation process, but is, rather, the result of a concentration dependent intermolecular aggregaiion. Variations in pH and temperature were found to affect both the rate and the extent of enzymic reactivation, whereas orthophosphate ions, ribonucleic acid, native ribonuclease, and low concentrations of B-mercaptoethanol had no effect. These findings are consistent with the hypothesis that the information necessary for the conversion of a newly synthesized polypeptide chain to the native form of the protein molecule resides in the amino acid sequence and does not involve a genetically determined template.