CHEMICAL BONDS IN NUCLEIC ACIDS 423 



more than 60% of niicleoside-5 '-phosphates from the ribonucleic acids of 

 yeast and calf liver, in addition to some free nucleosides and nucleoside 

 diphosphates. These latter substances, which are discussed later in connec- 

 tion with chain-branching, have one of their phosphate groups at the 5'- 

 position of the nucleoside residue. Thus there is good evidence that the 

 C 5 '-position is of major importance as an internucleotidic linkage point. 

 Further strong confirmatory evidence is to be found in the chemistry of 

 the oligonucleotides found in ribonuclease digests of ribonucleic acids. The 

 presence of 5'-phosphoester linkages in ribonucleic acids and the absence 

 of nucleoside-5 '-phosphates in chemical hydrolysates find an adequate 

 explanation in the theory of hydrolysis discussed above. ^^ 



5. Chemistry of Ribonuclease Action 



As clearly indicated, alkaline hydrolysis degrades ribonucleic acid to 

 mononucleotides without yielding larger fragments (oligonucleotides) con- 

 taining more than one nucleotide unit. Such larger fragments are of impor- 

 tance in the study both of nucleotide sequence and of the detail of the inter- 

 nucleotidic linkage. They can be obtained by the action of certain enzymes, 

 among which the most widely used is pancreatic ribonuclease. This enzyme 

 was obtained in crystalline form by Kunitz^* in 1940, and its use in recent 

 years has shed light on a number of important details of ribonucleic acid 

 structure. 



Allen and Eiler^ found that 0.25 equivalents of secondary phosphoryl 

 groups per atom of phosphorus were liberated during ribonuclease action, 

 and higher values (0.4-0.5) have since been reported ;*2'^^ no inorganic 

 phosphate was liberated. The enzyme, indeed, seemed to be a specific 

 type of diesterase and as such it gave both dialyzable and nondialyzable 

 fission products when allowed to act on ribonucleic acids. Weiner, Duggan, 

 and AUen^* found by titration that the ratios of monoesterified to diesterified 

 phosphate in the intact nucleic acid, and in the dialyzable and the non- 

 dialyzable fractions of the digests, were 1:10, 1:2, and 1:3, indicating that 

 considerable breakdown had occurred even in the nondialyzable fragments. 

 Analysis of the fractions obtained" showed that the smaller (dialyzable) 

 fragments were rich in pyrimidine nucleotide derivatives, while the larger 

 (nondialyzable) showed a high purine-pyrimidine ratio. These observa- 

 tions led to the suggestion"-^* that the enzyme acted at pyrimidine nucleo- 

 tide sites in the molecule. Evidence pointing in the same direction came 



S6M. Kunitz, J. Gen. Physiol. 24, 15 (1940). 



" E. Volkin and W. E. Cohn, J. Biol. Chem. 205, 767 (1953). 



" J. E. Bacher and F. W. Allen, J. Biol. Chem. 183, 633 (1950). 



98 See also G. Schmidt, R. Cubiles, B. H. Swartz, and S. J. Thannhauser, J. Biol. 



Chem. 170, 759 (1947) ; G. Schmidt, R. Cubiles, and S. J. Thannhauser, Cold Spring 



Harbor Symposia Quant. Biol. 12, 161 (1947). 



