HYDROLYSIS — ESTIMATION OF BASES IN PNA 193 



There is some question about the complete hberation of the purine bases 

 in 0.5 N HCl at 120° for 2 hours,^- '« but, in 1 A^ acid at 100° for 1 hour, 

 adenine and guanine are completely liberated as judged by the recovery 

 of adenine from adenylic acid (95 %) and of guanine from sodium guanylate 

 (100%) by paper chromatography^ or by the recovery of adenine from 

 adenylic acid after hydrolysis and precipitation as the silver salt (97%).^ 

 In 2 A^ H2SO4 at 100° purine nitrogen liberated from pancreas PNA reaches 

 a maximum in about 30 minutes, also indicating the complete liberation of 

 the purine bases from this type of PNA under these conditions.® The above- 

 mentioned experiments show that the purine bases are formed readily on 

 acid hydrolysis of PNA, without appreciable loss, either by paper chroma- 

 tography or by silver precipitation and without a significant amount of 

 deamination. Other experiments'^ in which an isotope dilution method was 

 used to estimate the purine concentration have indicated that as much as 

 7-8 % of both adenine and guanine may be destroyed by 1 A^ HCl at 100°. 

 The reasons for the apparent discrepancy between these results and those 

 mentioned above are not apparent. They probably depend in part on ex- 

 perimental technique and in part on experimental error. Direct evidence 

 that hydrolysis in 1 N HCl or H0SO4 at 100° for 1 hour liberates the purine 

 bases quantitatively is the recovery of from 98-99 % of the nitrogen of some 

 samples of yeast PNA as known purine and pyrimidine components.'^' ^ 



In agreement with the accepted theory that PNAs are polynucleotides 

 and that adenine and guanine are formed from adenylic and guanylic acids, 

 respectively, is the accompanying formation of reducing groups and inor- 

 ganic phosphate, as adenine and guanine are liberated during acid hydroly- 

 sis. The experiments of Levene and co-workers'^' " on the preparation of 

 a ribose phosphate from xanthylic acid and the inosinic acid derived from 

 yeast adenylic acid showed that the A^'-riboside linkage could be hydrolyzed 

 at pH 2 before appreciable amounts of inorganic phosphate were formed. 

 [Cf. Overend and Stacey, Chapter 2.] In 1 A^ or 2 A^ acid the liberation of 

 purine N likewise is slightly more rapid than the formation of inorganic 

 phosphate.® The methods which attempt to relate reducing sugar forma- 

 tion to adenine and guanine liberation involve the conversion of the ribose 

 formed to furfural and its distillation or estimation by colorimetry. [Cf. 

 Dische, Chapter 9.] The liberation of inorganic phosphate during acid hy- 

 drolysis of PNA has been used directly as a measure of the amount of purine 

 nucleotides present and indirectly as a measure of the acid-resistant pyrimi- 

 dine nucleotides.^'''' • ^* 



i» M. M. Daly, V. G. Allfrey, and A. E. Minsky, /. Gen. Physiol. 33, 497 (1950). 

 " R. Abrams, Arch. Biochem. and Biophys. 30, 44 (1951). 



12 P. A. Levene and A. Dmochowski, J. Biol. Chem. 93, 563 (1931). 



13 P. A. Levene and S. A. Harris, J. Biol. Chem. 95, 755 (1932); 98, 9 (1932); 101, 419 

 (1933). 



1^ W. Jones, J. Biol. Chem. 25, 87 (1916). 



