ION-EXCHANGE CHROMATOGRAPHY 



231 



GUANYLIC 

 5'o b 



01 02 01 0.2 



LITERS THROUGH COLUMN 



Fig. 15. Analysis of (a) enzymic, (b) alkaline digest of calf liver PNA by anion 

 exchange.^" 



Exchanger: Dowex-l -formate, 200-400 mesh, 7 cm. X 0.88 cm. 2. 

 Solutions: as shown. 



Sorbed material: (a) 100 mg. (20 ml.) PNA + 7 mg. ribonuclease digested 5 hr., 

 titrated with (112 /.leq.) NaOH to hold at pH 7.2; diluted to 25 ml.; 12 ml. + 0.8 

 ml. 0.1 M arsenate + 6 ml. (20 mg.) intestinal phosphatase; digested 90 min., ti- 

 trated with 1.5 ml. 0.05 M NaOH to hold at pH 8.5; diluted to 25 ml.; acidified to 

 pH 2.9 with formic acid ; centrif uged. Supernatant plus NH4OH sorbed on column, 

 (b) 4 ml. same PNA solution + 2 meq. NaOH; digested 15 hr. at 37°C.; diluted to 

 25 ml. with 2 meq. NH4 formate + 2 meq. NH4OH; sorbed on same column sub- 

 sequently. 



nucleosides, the adenosine appearing as inosine.'*' The recovery of these 

 components and the mode of isolation and discovery of the diphosphates 

 is shown in Fig. 16. 



The positions of the two diphosphates are of some interest. It might be 

 expected that the differentiation in ion-exchange behavior of these sub- 

 stances would depend on the bases to the same extent as in the monophos- 

 phates and thus bring about the same order of elution as is observed in the 

 monophosphates in pH ranges adjusted to offset the increased number of 

 acid groups. That such is the case is indicated by the order of the two 

 pyrimidine diphosphates in Fig. 16 and by the fact that adenosine-5'- 

 pyrophosphate (ADP) comes between them (see Fig. 17, in which the peak 

 positions of several substances examined independently are indicated by 

 arrows). The position of inorganic pyrophosphate between ADP and uridine 

 diphosphate is also similar to orthosphosphate in the monophosphate series. 

 It is also apparent that the diphosphates as a group are sufficiently strongly 

 bound to the anion-exchange resin to follow the last of the monophosphates, 

 overlapping only slightly with guanylic (and inosinic) acid. (The behavior 



