Formation of Randomly Paired Disulfide Bonds in Des-(121–124)-ribonuclease after Reduction and Reoxidation

Abstract

The enzymically inactive des-(121–124)-ribonuclease was prepared by limited peptic digestion of bovine pancreatic ribonuclease after the modification of the original method of Christian B. Anfinsen. The circular dichroism, at 210 to 300 mµ of des-(121–124)-ribonuclease, is similar to that of intact ribonuclease with the exception that lower negative circular dichroism around 220 mµ was observed with the former. When reduced des-(121–124)-ribonuclease was completely oxidized by air at a concentration of approximately 0.02 mg of protein per ml, the reoxidized des-(121–124)-ribonuclease was predominantly in the monomeric form. However, the circular dichroism at 210 to 300 mµ of the reoxidized des-(121–124)-ribonuclease was featureless and entirely different from the original pattern, indicating a disordered structure. Analysis by gel filtration of size distribution of cross-linked fragment complexes produced from the reduced-reoxidized des-(121–124)-ribonuclease by BrCN cleavage also indicated the randomly paired disulfide bonds. The results are interpreted to show that the removal of the 4 residues (-Asp-Ala-Ser-Val) from the carboxyl end of the amino acid sequence of reduced ribonuclease causes destruction of the information to form correct disulfide bonds upon oxidation. Thus, it is concluded that the native conformation of bovine pancreatic ribonuclease cannot be formed during biosynthesis until the polypeptide chain has been extended beyond residue 120. The results are consistent with the previously reported observations on an inactive disordered fragment of residues 1 to 126 obtained from staphylococcal nuclease (149 residues).