204 HUBERT S. LORING 



funnel to remove stray particles of the flocculent silver-purines and set aside for anal- 

 ysis of the p3'rimidine components. 



The silver-purine precipitate is subjected to four successive treatments with 10-ml. 

 portions of 0.1 N HCl to extract the free purine bases. In each case the suspension of 

 silver salts in the acid is heated in a boiling water bath for 5 minutes with occasional 

 stirring and then allowed to cool for 10 minutes. The solids are sedimented by cen- 

 trifugation, and the supernatant liquid is poured onto the funnel used in the separa- 

 tion of the purine and pyrimidine fractions. The acid solution is allowed to remain 

 in the funnel for several minutes in contact with a few particles of silver-purine re- 

 tained in the previous step before suction is applied. The combined filtrates from this 

 procedure, constituting the pure fraction, are diluted with 0.1 A'^ HCl to 50 ml. A 

 10-ml. aliquot diluted to 100 ml. with 0.1 N HCl is used for optical density measure- 

 ments at 240, 252, 262, 276, and 280 m^. The adenine, guanine, and total purine con- 

 centrations are calculated from the optical densities as previously described. 



The pyrimidine fraction is warmed to about 70°, 3 ml. of 1 iV HCl added, and the 

 AgCl precipitate allowed to coagulate and removed by filtration. The precipitate is 

 washed three times by suspension in 5-ml. portions of 0.1 N HCl. The combined fil- 

 trates are diluted to about 45 ml., the pH of the solution adjusted to 4.7 with NaOH 

 (pH meter), and the volume brought to 50 ml. Two 5-ml. aliquots are treated with 



I ml. of a filtered phosphatase solution containing 1 mg. of enzyme'' and the result- 

 ing solutions are incubated at 38° for 3 hours to bring about dephosphorylation of 

 the pyrimidine nucleotides. One aliquot, diluted to 100 ml. with 0.1 N HCl, is used 

 for optical density measurements at 260, 267, 278, and 295 m/x. Cytidine, uridine, and 

 total pyrimidine concentrations were evaluated as previouslj' described and corrected 

 for 3.5% deamination of cytidylic acid. The values obtained in this way may be 

 slightly high if small amounts of ultraviolet-absorbing amino acids were present 

 initially. The latter may be removed as follows: A second aliquot is adjusted to 

 pH 8.3 with NaOH, the solution filtered through a Dowex 1 (bicarbonate) column 

 (2 cm. X 3 sq.cm.), and the resin washed with about 70 ml. of 2% NaHCOs. The pH of 

 the combined effluent is carefully adjusted to 1.0 with H2SO4 and the volume to 

 100 ml. Optical density measurements are made and the concentrations of cytidine, 

 uridine, and total pyrimidine nucleoside calculated as previously described. 



4. Application to Purified Yeast Ribonucleic Acids 



The results of the application of the above mentioned analytical pro- 

 cedures to different purified samples of yeast PNA are summarized in Table 



II in comparison with those of several other investigators. [Cf. Magasanik, 

 Chapter 11.] They show that from 97 to 99 % of the nitrogen of commercial 

 sodium ribonucleate or of a carefully prepared nucleic acid sample can be 

 accounted for in terms of known purine and pyrimidine components. Simi- 

 lar almost complete recoveries of the nitrogen of other yeast ribonucleic 

 acid samples are reported in several instances by other workers.^' 24. 25, ss. 59 

 In relation to phosphate content the results show that purine and pyrimi- 

 dine bases occur in very nearly equimolar quantities with phosphorus and 

 confirm the general opinion that ribonucleic acids are essentially poly- 

 ps A. Deutsch, R. Zuckerman, and M. S. Dunn, Ind. Eng. Chem., Anal Ed. 24, 1769 



(1952). 

 " G. W. Crosbie, R. M. Smellie, and J. N. Davidson, Biochem. J. 54, 287 (1953). 



