The Isolation of Mononucleotides after Hydrolysis of Ribonucleic Acid by Crystalline Ribonuclease* by Hubert

Since the discovery by Jones (1) of a thermostable enzyme in the pancreas, capable of hydrolyzing ribonucleic acid without the release of either phosphoric acid or purine bases, the evidence as to the nature of its action has been extremely conflicting. Jones and Perkins isolated four mononucleotides from enzyme-treated nucleic acid and concluded that the action of the enzyme consisted in breaking nucleotide linkages only (2). Levene, however, was not successful in his attempts to repeat the experiments of Jones ((3) p. 312) and in a paper with Schmidt (4) reached the conclusion that “The function of the enzyme is that of a depolymerizing agent, limited to the dissociation of the tetranucleotides of high molecular weight into those of lower molecular weight.” A thermostable enzyme with properties identical with those of the impure extracts mentioned above was isolated and crystallized by Kunitz and provisionally named ribonuclease (5). Its action consisted in the liberation of free acid groups without the formation of free phosphoric acid, and in the formation of split-products not precipitable by glacial acetic acid and readily diffusible through collodion or cellophane membranes. Allen and Eiler confirmed the crystallization of the enzyme and showed that the increase in free acid groups after enzymic action approached 1 equivalent for each mole of ribonucleic acid used, assuming a value of 1286 for the molecular weight of ribonucleic acid (6). Subsequently Bolomey and Allen showed that the hydrolytic action of a non-specific phosphatase was 50 to 150 per cent greater on ribonucleic acid that had been treated previously with ribonuclease in comparison with the untreated acid (7). In the present paper me report the isolation and identification of four nucleotides from ribonucleic acid treated with crystalline ribonuclease, thus confirming the original finding of Jones. Control experiments on the fractionation of ribonucleic acid by similar procedures in the absence of enzyme treatment gave amorphous products with the general properties of the original nucleic acid.