HYDROLYSIS — ESTIMATION OF BASES IN PNA 199 



c. Liberation of Acid Groups 



The earlier experiments mentioned above were concerned primarily with 

 the preparation of the nucleotides in pure form and with their chemistry. 

 It is likely that appreciable amounts of inorganic phosphate, nucleosides, 

 and deaminated products as well as nucleotides were formed under the 

 more severe conditions employed. When a 2 % solution of sodium nucleate 

 in 0.1 N NaOH is heated at 100°, there is at first a rapid liberation of acid 

 groups^^' ^'^ followed by a slower liberation of acid until a maximum value 

 is reached corresponding to about 1.08 equivalents per mole of nucleic acid 

 P. As no increase in inorganic phosphate was found under these conditions, 

 it is evident that the acid formed was due to the hydrolysis of phosphate 

 ester linkages in the original nucleic acid structure. Of interest is the fact 

 that the amount of acid formed, 1.08 equivalents per mole of P, is greater 

 by approximately 8 % than that expected from a nucleotide polymer con- 

 taining only diester linkages. The extent to which deamination of purine 

 nucleotides occurs under these conditions has not been determined and is 

 probably negligible. Cytidylic acid is deaminated to the extent of 2 and 12 % 

 in 0.01 A'' and 0.1 A^ NaOH, respectively, but is not significantly affected 

 at pH ll.-*' Appreciable deamination, from 10% to 33%, apparently occurs 

 in 1 N alkali at 37°.^! 



II. Estimation of Purine and Pyrimidine Components in PNA" 



1. Chemical Fractionation of Purine Bases and Pyrimidine 

 Nucleotides and Their Estimation by Absorption 

 Spectrophotometry 



As discussed above, hydrolysis of PNA with 1 A'' acid at 100° for 1 hour 

 leads to the formation of a mixture of the purine bases and pyrimidine 

 nucleotides with small amounts of pyrimidine nucleosides and possibly oxy- 

 purines. As the purine bases form highly insoluble silver salts in acid solu- 

 tion^^' ^2' ^^ in contrast to either pyrimidine nucleotides or nucleosides, it is 

 possible to effect a relatively quantitative separation of the two types of 

 components by this procedure. If the purine bases are redissolved in dilute 

 HCl and^ the pyrimidine nucleotide fraction, after removal of silver ions, 

 treated with prostatic phosphatase, two relatively simple binary mixtures 



*' H. S. Loring, H. W. Bortner, L. W. Levy, and M. L. Hammell, J. Biol. Chem. 196, 



807 (1952). 

 *" H. W. Bortner, Dissertation, submitted to Stanford University for the Degree of 



Doctor of Philosophy in Chemistry, 1952. 

 " D. H. Marrian, V. L. Spicer, M. E.Balis, and G. B. Brown, J. Biol. Cheyn. 189, 533 



(1951). 

 " R. Feulgen, Z. phjsiol. Chem. 102, 244 (1918). 

 " S. E. Kerr and K. Seraidarian, J. Biol. Chem. 159, 211 (1945). 



