A simplified procedure for the preparation of cytidylic acid and diammonium uridylate from yeast nucleic acid.

Previously published methods for the preparation of the pyrimidine nucleotides are relatively involved procedures requiring either repeated crystallization of the brucine salts (1) or fractionation of the free acids by their differential solubility in pyridme (2). Although the yields obtained by the former method were not published, we have isolated less than 2 per cent of the original nucleic acid as crystalline diammonium uridylate by this procedure. Yields of 5.6 and 4 per cent of the original nucleic acid as crystalline cytidylic and “nearly” crystalline uridylic acids respectively were reported for the pyridine extraction procedure. Other workers, however, have found lower yields by the latter method (3) and in our own laboratory the yield of crystalline cytidylic acid isolated from a sample of yeast nucleic acid by the pyridine method was 1.2 per cent. Although the results of bioassays far cytidylic and uridylic acids indicate that the concentrations of these constituents in ribonucleic acid are less than those expected from a tetranucleotide structure (4), it is evident from the small yields mentioned that the isolation procedures are highly inefficient. The extent to which nucleosides or nucleotides are produced by alkaline hydrolysis of yeast nucleic acid depends on the concentration and type of alkali used and on the temperature at which the hydrolysis is carried out (1, 5). The published procedures for the preparation of nucleotides have avoided hydrolysis to nucleosides by the use of ammonia at 115-120” in an autoclave (5) or alternately by the use of stronger alkali at room temperatures for relatively long periods of time (6, 7). In view of the difficulties inherent in the former procedure and the uncertainty regarding the extent of hydrolysis in the latter, it seemed desirable to find conditions in which nucleic acid could be converted to nucleotides by refluxing in the presence of a relatively strong base. Because of the insoluble nature of some of the barium salts of the nucleotides (8,9), the use of barium hydroxide as a hydrolyzing agent was studied. In order to find optimum conditions