230 



WALDO E. COHN 



LITERS THROUGH COLUMN 



Fig. 14. Separation of the isomeric 2', 3', and 5' nucleotides of ribonucleic acid by 

 anion exchange in a formate system. '^ 



Exchanger: Dowex-l(X 2%) -formate, 400 mesh, 5 cm. X 0.82 cm.^. 

 Solution: Formic acid, ammonium formate as shown. 



Sorbed material : Combined alkaline digest and 5' nucleotides (diesterase digest) 

 prepared as follows: 25 mg. calf liver PNA + 1 ml. 0.5 A^ NaOH, 37°C., 18 hr.; 

 diluted to 50 ml.; added 2 mg. each of 5' nucleotides (prepared as described in 

 Fig. 16) + 0.5 meq. NH4 formate + NH4OH to 0.1 M. 



(7) Application to Digests. A demonstration of the separation of the 5', 

 2', and 3' isomers of all four ribonucleotides is shown in Fig. 14. ^^ It has 

 been customary, in order to save time, to double the concentration of for- 

 mate and lower the pH with formic acid after removal of the uridylic acids 

 (see Fig. 15). 



The use of this sytem in the analysis of unknown digests and of the usual 

 alkaline digest is shown in Fig. 15.'*'' This is the actual experiment in which 

 the 5' nucleotides were first isolated from an enzyme digest of PNA (See 

 also Cohn and Volkin^^ and Fig. 16). 



2. POLYPHOSPHONUCLEOSIDES 



a. Diphosphates 



The mixed 2', 5'-, and 3',5'-diphosphates of cytidine and uridine have 

 been recovered by ion exchange from digests of PNA with rattlesnake 

 venom freed of 5' monoesterase.^^- ^^ These digests also contain the 5' 

 nucleotides of all four bases, and 3' pyrimidine nucleotides, and all four 



« W. E. Cohn and E. Volkin, Arch. Biochem. and Biophys. 35, 465 (1952). 

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



