194 HUBERT S. LORING 



6. Liberation of Inorganic Phosphate and Formation of Pyrimidine Nucleotides 



The remarkable stability of the pyrimidine ribonucleotides to mild acid 

 hydrolysis, in contrast to the purine components, was first noted by Levene 

 and Jacobs in experiments in which the mixed nucleotides were prepared 

 as barium salts after a 2-hour hydrolysis of yeast PNA with 2 % H2SO4 .^^• 

 ^®(*^ Subsequently, after the discovery of nucleotides as products of alkaline 

 PNA hydrolysis, the mixed pyrimidine nucleotides produced by acid hy- 

 drolysis were fractionated into crystalline barium and brucine uridylates 

 and into free cytidylic and uridylic acids. ^^- ^* That the phosphoric acid of 

 the pyrimidine components is slowly hydrolyzed by acid was shown by the 

 preparation of cytidine and uridine after acid hydrolysis of the nucleotides^^ 

 and by experiments on the rate of liberation of inorganic phosphate from 

 yeast FNA^^^)- " in 5 % H2SO4 at 100°. Jones showed that an amount of in- 

 organic phosphate corresponding to 53.9 % of that present in the nucleic 

 acid was liberated during the first 2 hours, but that the amount formed sub- 

 sequently corresponded to a rate of only 10 mg. of magnesium ammonium 

 phosphate (1.26 mg. P) per gram of nucleic acid per hour. Assuming that 

 the relatively labile phosphate corresponded to that bound to purine nu- 

 cleosides and correcting the 2-hour period for inorganic phosphate formed 

 from the pyrimidine components, Jones calculated that approximately 50 % 

 (50.7 %) of the nucleic acid phosphate was bound to the purine nucleosides 

 and a similar quantity to the pyrimidine nucleosides in yeast PNA. This 

 occurrence of labile and stable phosphate in nearly equal proportions in 

 yeast PNA has been largely responsible for the tetranucleotide concept of 

 PNA structure.isC') In 2 N H2SO4 after 1 hour at 100° Kerr et aU have 

 found a slightly higher value for inorganic phosphate formed from yeast 

 PNA, namely about 57-58 %. The rates of phosphate liberation from pure 

 cytidylic and uridylic acids is appreciably higher than the value given 

 by Jones, but the agreement between different investigators is only fair, 

 e.g., approximately 9% for both nucleotides after 1 hour in 0.1 A^ H2SO4 at 

 ]^00°i9a,b g^g compared with 13.8% for cytidylic acid under the same condi- 

 tions or with 15.5% in 1 AT acid.^" The recovery of purine and pyrimidine 



16 P. A. Levene and W. A. Jacobs, Ber. 44, 1027 (1911). 



" P. A. Levene and L. W. Bass, "Nucleic Acids," ACS Monograph Series. The 



Chenucal Catalogue Co., New York, 1931 : (a) p. 221 ; (b) p. 274; (c) p. 57; (d) p. 193; 



(e) p. 265. 

 " P. A. Levene, Proc. Soc. Exptl. Biol. Med. 15, 21 (1917). 

 18 S. J. Thannhauser and G. Dorfmuller, Z. physiol. Chem. 100, 121 (1917); 104, 65 



(1919); Ber. 51,467 (1918). 

 19* A. M. Michelson and A. R. Todd, /. Chem. Soc. 1949, 2476. 

 i^b G. R. Barker, J. M. Gulland, H. Smith, and J. F. Thomas, /. Chem. Soc. 1949, 



904, find an average value of 7% dephosphorylation for disodium uridylate, [a]^ = 



19.8° (anhydrous) in 0.1 N H2SO4 at 100°. 

 2» P. A. Levene and E. Jorpes, /. Biol. Chem. 81, 575 (1929). 



