ION-EXCHANGE CHROMATOGRAPHY 235 



formed by brief acid hydrolysis. Either system (increasing NaCl at constant 

 pH or increasing HCl) can be adapted to gradient development and other 

 anions (e.g., formate) can be used, as has been demonstrated for the acid- 

 soluble polyphosphates.^' 



It is of some interest to compare the relative positions of the polynu- 

 cleotides in the elution sequence at pH 2 in a chloride system with those 

 predicted from the positions of the mononucleotides at this pH. Polynu- 

 cleotides ending in C3d:idylic acid (e.g., AC and GC) precede those ending 

 in uridylic acid, just as cytidylic acid itself precedes uridylic acid. The 

 nature of the purine nucleotide attached to the end pyrimidine nucleotide 

 exerts an influence predictable from its own behavior, but secondary in 

 importance. Thus, AC precedes GC, AGC precedes GGC, and AAGC 

 precedes AGGC. From the observation that GAC precedes AGC, it would 

 appear that the third nucleotide exerts still less influence than the second 

 one. It should also be noted that the number of phosphate groups exerts 

 the expected influence; thus AC precedes AAC and GU precedes GGU. 

 Finally, it may be remarked that the elimination of the terminal (second- 

 ary) phosphoryl group from a dinucleotide makes the residual dinucleoside 

 monophosphate behave like a mononucleotide. Thus, dephosphorylated 

 GC appears in the cyiidylic acid region. The behavior of a dephosphorylated 

 trinucleotide has not been investigated. 



c. The Deoxyrihonuclease Digest 



The results of Sinsheimer,''^ obtained on the deoxyrihonuclease digest of 

 DNA at pH 4-5.5 in chloride and acetate systems, are in general agreement 

 with the above. The order of elution of dinucleotides of cytidylic acid is 

 CC, TC (plus CT), AC, and GC (plus CG). From his earlier studies,^^ it is 

 known that the order of deoxjonononucleotides in this pH range is C, T, 

 A, and G. In the trinucleotide region, the order is CCC, CCT, and CTT; 

 these precede GG. In every case where one cytidylic acid residue is replaced 

 by 5-methylcytidylic acid, the polynucleotide is advanced in position (it 

 will be recalled, as in Fig. 11, that 5-methylcytidylic acid precedes cyti- 

 dylic). Thus MCC precedes CCC, MCT precedes CCT, etc. 



IV. Separations Involving Sugar-Borate Complexing 



1. Sugars (Bor.\te Exchanger and Solution) 



Although the sugars have acid ionization properties which could con- 

 ceivably be used in ion-exchange manipulations, these constants lie so far 

 on the alkaline side (ca. 13) as to be relatively unattractive or unmanage- 

 able. However, just as the use of complex-forming acids rendered the 



" H. Schmitz, R. B. Hurlbert, and V. R. Potter, J. Biol. Chem. 209, 41 (1954). 



