FRANK M. HUENNEKENS 



(similarly for other purine and pyrimidine nucleotides), panto- 

 thenic-2,4-phosphate, FAD-X (a cyclic derivative of FAD), and 

 riboflavin-4', 5 '-phosphate, all cyclic phosphates, are formed 

 during the degradation of uridine diphosphate glucose, nucleic 

 acids, CoA, and FAD, respectively. Such cyclic phosphates 

 may also appear and play a role during enzymatic processes, for 

 these substances are powerful phosphorylating agents. 



With the advent of methods for obtaining coenzymes 

 largely free from impurities, especially of similar structure, it has 

 become possible to achieve final and conclusive purification via 

 crystallization — a finding which has helped to restore nucleo- 

 tides to their rightful place amongst respectable organic com- 

 pounds. Occasionally, it has been necessary to employ a crutch 

 for this process by crystallizing the nucleotide as a complex with 

 some organic base such as acridine, quinine, or cyclohexyl- 

 amine. 



Pure coenzymes, if isolated in sufficient quantity, can be 

 subjected to structure determination both by the methods of 

 classical organic chemistry and by certain sensitive, small- 

 scale methods suggested by enzymology itself. The coenzymes 

 usually have no melting points, owing to their saltlike character, 

 and elemental analysis is usually reserved for confirmatory 

 evidence. However, degradation in acid or basic media, and 

 subsequent examination of the liberated fragments, can provide 

 a great deal of information. Small quantities of the fragments 

 can be detected and compared with authentic compounds by 

 means of paper chromatography. Tremendous ingenuity has 

 been exercised in the composition of solvent systems for separat- 

 ing closely related compounds and in devising spray reagents or 

 other means for locating these compounds on paper. A clas- 

 sical example of the potentialities of this technique has been 

 afforded by the work of Baddiley and Thain (3) who obtained 

 decisive evidence on the structure of CoA, although only minute 

 amounts of the partially purified coenzyme were available. 



Finally, it is extremely fortunate if the derived fragments can 

 be estimated enzymatically, for example, AMP by a specific 



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