586 G. SCHMIDT 



determination or, in the case of the phosphodiesters of nitrophenols which 

 are practically colorless, by direct photometric determination of the yellow 

 color of the liberated nitrophenol. 



Monophenyl phosphate, p-nitrophenyl phosphate, the glycerophosphates 

 and all mononucleotides are resistant to the enzyme. 



Nature of Degradation Products. Incubation of PNA as well as of deoxy- 

 ribonuclease I digests of DNA results in the cleavage of practically all 

 internucleotide bonds. (The appearance of small amounts of inorganic 

 phosphate in many digests originates most likely from small contamina- 

 tions of the phosphodiesterase preparations with phosphomonoesterases.) 

 The action of snake venom phosphodiesterase on PNA was first studied by 

 GuUand and Jackson,^'* who found that such digests contained phosphoryl 

 groups which were hydrolyzed by a specific 5 '-nucleotidase (see below) 

 and concluded that a considerable amount of 5 '-phosphoryl groups must 

 be present in PNA itself. The action of this enzyme on PNA was con- 

 vincingly elucidated in the investigations of Cohn and Volkin.^^^ In the 

 case of deoxyribooligonucleotides, the final hydrolysis products are exclu- 

 sively the four 5'-mononucleotides. The exhaustive digestion of PNA with 

 snake venom phosphodiesterase results in a mixture of the four 5 '-nucleo- 

 tides, of pyrimidine-2',5'- and -3',5'-diphosphomononucleotides, and of 

 an amount of purine nucleosides approximately equivalent to that of the 

 diphosphonucleotides. 



Thus, in contrast to the action of ribonuclease, that of snake venom 

 diesterase causes specifically the cleavage of the 3 '-ester linkages between 

 the phosphoryl groups and the corresponding nucleoside residues. The 

 structural implications of these observations have been discussed in Chap- 

 ter 12. 



The practically complete cleavage of PNA and of all deoxyribooligo- 

 nucleotides to mononucleotides or — in the case of PNA — to mixtures of 

 mononucleotides and mononucleotide phosphate esters leads to the con- 

 clusion that all internucleotide bonds in these polynucleotides regardless 

 of their structural differences are cleaved by snake venom phosphodieste- 

 rase. However, this general statement requires one qualification: Only 

 those compounds are hydrolyzed by the enzyme which do not have a sec- 

 ondary phosphoryl group in the 3'-position. Thus, dinucleotides of the type 

 shown in the lower part of Figure 8 are resistant to the enzyme, but nu- 

 cleotide-nucleoside esters originating from dinucleotides by the removal 

 of the terminal phosphoryl group by a phosphomonoesterase are hydrolyzed 

 by snake venom diesterase. 



Consequently, all oligonucleotides of deoxyribonuclease I-digests are 



1" J. M. Gulland and E. M. Jackson, Biochem. J. 32, 597 (1938). 

 i'" W. E. Cohn and E. Volkin, /. Biol. Chem. 203, 319 (1953). 



