A Practical Preparation of Ribonuclease-S and the Action of Subtilisin on Ribonuclease-B ‡

All preparations of ribonuclease-S so far reported have employed limited digestion of ribonuclease-A with subtilisin,' a bacterial proteinase from B. subtilis described by Giintelberg and Ottesen.' The original supply of this latter enzyme is now exhausted. Another sample of a crystalline proteinase from B. subtilis (NOVO Enzyme) has been shown to differ from the original in several respects,' including its ability to produce satisfactory yields of ribonuclease-S (ref. 1, footnote 2). This paper reports the successful use of a commercially available bacterial proteinase ("Nagarse") for the digestion of ribonuclease and the detailed procedures found most useful for the preparation of the RNase-S.t in gram amounts. Some incidental observations on the action of subtilisin on ribonuclease-B are also included. A number of commercial samples of crystalline bovine pancreatic ribonuclease have been used in this work: Armour Inc., Chicago, Ill. Fellow during the period of this study. National Science Foundation. ¶ The abbreviations used are: RNase-A, the principal chromatographic component of bovine pancreatic ribonuclease; RNase-S, subtilisin-modified ribonuclease; S-Peptide, the 20 residue peptide component obtained from RNase-S; S-Protein, the protein component obtained from RNase-S; RNase-B (-B1,-B2), the minor chromato-graphic components of native ribonuclease; RNase-S (B.) or RNase-S (B2), the enzyme obtained by subtilisin modification of the indicated native B component.