The preparation of subtilisn-modified ribonuclease and the separation of the peptide and protein components.

The proteolysis of bovine pancreatic ribonuclease by subtilisin has been shown to involve the formation of an enzymically active intermediate which is further degraded to inactive products (1, 2). This paper describes the preparation of the modified active ribonuclease, RNase (S)l and its fractionation into two components, a peptide (S-peptide) and a protein (S-protein). Neither S-peptide nor S-protein alone shows appreciable ribonuclease activity. When these components are mixed in equimolar proportions, almost the full enzymic activity is recovered. The only observed change in covalent structure during the conversion of RNase A to RNase S is the hydrolysis of the peptide bond between residues 20 and 21 as numbered from the NH&erminal end of the single chain of RNase A. A comparison of the enzymic properties of RNase A, RNase S and RNase S’ (the reconstituted enzyme) under a number of conditions is presented. A brief report of some of these observations has been published (3).