HYDROLYSIS — ESTIMATION OF BASES IN PNA 195 



components in most of the recently published analyses lies within the range 

 expected from the above mentioned values, viz., 0.50-0.57 and 0.36-0.46 

 moles per mole of P for purines and pyrimidines, respectively. [Cf. Maga- 

 sanik, Chapter 11.] 



In estimating the amounts of pyrimidine nucleotides in PNA after acid 

 hydrolysis, it is apparent from the above discussion that appreciable error 

 may result if hydrolysis to nucleosides is neglected .^^' ^- Of significance also 

 is the possible deamination or destruction of the pyrimidine compounds. 

 In 0.4 N sulfuric acid at 100° a 2 % deamination of cytidine per hour and 

 in 1 AT" HCl a 3-4 % deamination of cytidylic acid have been reported.^^- ^' 



c. Liberation of Pyrimidine Bases 



The hydrolysis of the pyrimidine components to the free pyrimidine 

 bases presents considerable difficulty. Procedures for the preparation of 

 cytosine and uracil from yeast PNA involve a 2-hour hydrolysis at 175° in 

 25 % H2SO4 .^^f=> Under these conditions there is a considerable deamination 

 of cytosine to uracil, a fact which in the earlier literature placed doubt on 

 the existence of uracil as a nucleic acid component. More recently the lib- 

 eration of the free pyrimidine bases from PNA by 0.4-6 N HCl at 120°,^ by 

 concentrated formic acid (98-100 %) and by 20 % HCl at 175°," and by 12 

 N perchloric acid-^ has been studied. The results with 0.4-6 A^ HCl at 120° 

 show an increasing liberation of pyrimidine bases as acid concentration is 

 increased, but the yields of cytosine relative to uracil are low, indicating a 

 considerable degree of deamination under these conditions. In concentrated 

 formic acid cytosine is apparently preserved from deamination to uracil, 

 but the recovery of pyrimidine bases in yeast PNA relative to P amounted 

 to only 0.327 mole per mole, in contrast to values as high as 0.46 found by 

 other methods after 1 N acid or alkaline hydrolysis.^' *• " Hydrolysis of 

 yeast PNA with 12 A^ perchloric acid^" for 1 hour at 100° causes no appreci- 

 able destruction of either adenine, guanine, cytosine, uracil, or thymine, 

 but the recovery of total pyrimidine base when this procedure was applied 

 to yeast PNA, 0.37 moles per mole P, again indicates an incomplete libera- 

 tion from PNA. At the present time, therefore, under the conditions studied 

 neither 20% HCl, concentrated formic acid, nor 12 N perchloric acid can 

 be said to lead to the quantitative liberation of the pyrimidine components 

 of PNA in the form of free bases. 



21 21. R. Markham and J. D. Smith, Biochem. J. 49, 401 (1951). 



" H. S. Loring, J. L. Fairley, H. W. Bortner, and H. L. Seagran, /. Biol. Chem. 197, 



809 (1952). 

 " H. S. Loring, and J. Mc T. Ploeser, /. Biol. Chem. 178, 439 (1949). 

 " A. Marshak and H. J. Vogel, J. Biol. Chem. 189, 597 (1951). 

 26 E. Chargaff, B. Magasanik, E. Vischer, C. Green, R. Doniger, and D. Elson, J . 



Biol. Chem. 186,51 (1950). 



