ON THE ENZYMIC ACTIVITY OF SUBTILISIN-MODIFIED RIBONUCLEASE

Abstract

The initial stage of the proteolysis of bovine pancreatic ribonuclease (RNase) by subtilisin results in the production of a modified RNase molecule whose enzymic activity is very similar to that of the native material." 2 The presence of this altered form of the enzyme (RNase-S) is easily demonstrated by its chromato- graphic behavior on the Amberlite resin IRC-50, on which it moves as a discrete peak clearly separated from and following the major component (RNase-A) of the native enzyme. The finding of two N-terminal amino acid residues and the separation of two peptides from the performic acid-oxidized material indicate that the single peptide chain of RNase-A has been cleaved at one bond and that the two parts remain associated in RNase-S. These original observations have now been confirmed and extended. Subtilisin appears to attack first the alanyl-seryl bond between residues Nos. 20 and 21, counting from the N-terminal end of the RNase-A chain. By careful treat- ment with trichloroacetic acid it has been possible to separate this N-terminal pep- tide (RNase-S-Pep) from the rest of the molecule (RNase-S-Prot). These fractions have only traces of residual enzymic activity. On mixing a solution of the trichloro- acetic acid precipitate with an equivalent amount of the supernatant fluid, the full enzymic activity of the unfractionated material is regenerated.