On the preparation of bovine pancreatic ribonuclease A.

Many studies on ribonuclease would benefit if reproducible, purified samples of the enzyme were readily available. The present report summarizes experiments on the preparation and storage of chromatographically purified bovine pancreatic ribonuclease A.’ Much of the earlier work on ribonuclease was done with a single, large, commercial batch of material (Armour Lot 381059) which was isolated from beef pancreas by salt and solvent precipitation methods patterned after those of Kunitz (1) and McDonald (2). When first examined chromatographically on columns of IRC-50 in 1955 (3), over 90% of this sample consisted of a single component, ribonuclease A. By 1959, however, the lot was nearly exhausted and had deteriorated somewhat, for King and Craig (4) obtained only a 70% yield of ribonuclease A by countercurrent distribution, and a similar yield was observed on chromatography. To supply our immediate needs for purified enzyme, ribonuclease A was isolated by the procedure of Hirs, Moore, and Stein (5), with commercial samples as starting material. It was found that preparations so obtained were often inhomogeneous. Changes had occurred subsequent to the chromatographic step and had progressed during storage. The reversible formation of active aggregates upon lyophilization has been reported elsewhere (6). These several observations prompted the experiments that are summarized in this communication. The development of a rapid chromatographic technique for the evaluation of the homogeneity of samples of the enzyme has been an integral part of the present research. Protein prepared by the procedures herein described has been the starting material for studies on the reduction and carboxymethylation of the enzyme which are reported in the accompanying paper (7).