ION-EXCHANGE CHROMATOGRAPHY 227 



TABLE II 

 pK Values of Isomeric Nucleotides and Related Substances 



Adenine 

 Adenosine 



Adenosine-5 '-phosphate 

 Adenosine-2 '-phosphate 

 Adenosine-3 '-phosphate 

 H3PO4 



/3-Glycerophosphate 

 Glucose-1 -phosphate 

 Cytosine 

 5-Methylcytosine 

 Cytidine 



5-Methylcytidylic acid 

 Cytidine-5' -phosphate 

 Cytidine-2' -phosphate 

 Cytidine-3' -phosphate 



" Alberty et alM (25°, 0.15 M NaCl). 



" Kuna, M., U.S. Atomic Energy Comm. Rept. ORNL-318 (May 10, 1949); quoted by Cohn." 



"^ Cavalieri.35 



'' Wassermeyer'* (see Levene and Bass," p. 231). 



* Levene and Simms" (see Levene and Bass,'^ pp. 223, 228). 

 / Shugar and Fox" (spectrophotometric). 



" Cohn'3 (spectrophotometric). 



* Fox el a!.'* (spectrophotometric). 

 ' Deoxy compound. 



' Loring et al.'^ 



* For deoxy 5' nucleotides: R. O. Hurst, A. M. Marko, and G. C. Butler, J. Biol. Chem. 204, 842 (1953) 



higher is necessary for their separation. Formate and acetate are more 

 manageable from the standpoint of pH control and buffer capacity in this 

 range and give good separations of the 5'-, 2'-, and 3'-phosphates of cytidine, 

 in that order. Attempts to prepare large amounts with 0.05-0.1 M formic 

 acid have given incomplete separation, apparently owing to an overloading 

 of the columns as well as to an excessively rapid elution with the higher acid 

 concentration.^^ Deoxycytidylic acid (also a 5' ester) is inseparable from 

 the ribose analogue under these conditions, but can be separated Avith the 

 aid of borate complexing (see Sect. IV.2.c). Deoxy-5-methylcytidylic acid 

 precedes the 5' nucleotide(s) by a small margin. Fig. lla^^ demonstrates 

 the separation of these substances with acetate buffers. This experiment is 

 similar to that in which were discovered the existence of 5-methylcytidylic 

 acid^^ in deoxycytidyhc acid and the existence of tw^o isomers (2' and 3') 



38 J. J. Fox, L. F. Cavalieri, and N. Chang, J. Am. Chem. Soc. 75, 4315 (1953). 



" H. S. Loring, H. W. Bortner, L. W. Levy, and M. L. Hammell, /. Biol. Chem. 



196, 807 (1952). 

 " W. E. Cohn and E. Volkin, Nature 167, 483 (1951). 

 « W. E. Cohn, /. Am. Chem. Soc. 73, 1539 (1951). 



