NICOTINAMIDE NUCLEOTIDE ENZYMES 121 



by codehydrogenase II, Robison ester apodehydrogenase (Zwischen- 

 ferment), and riboflavin enzyme. If the codehydrogenase II is the 

 speed-hmiting factor in this system, the oxygen uptake is a measure 

 of its concentration (5). It is probable that the old yellow enzyme 

 used by Warburg and Christian is an artifact. The cytochrome c 

 reductase of Haas, Horecker, and Hogness (38) seems to be the 

 natural acceptor for the hydrogen of the reduced codehydrogen- 

 ase II. 



These methods of determination have been used not only to 

 elaborate the purification of the codehydrogenases but also to study 

 their distribution from the standpoint of vitamin research. The 

 codehydrogenases occur in an equilibrium: coenzyme ^ dihydroco- 

 enzyme. Since the stabilities of the oxidized and the reduced forms 

 are different (see Table 4), the proportions of coenzyme and dihy- 

 drocoenzyme can be determined by heat extraction with acid or 

 with alkali, according to Adler and Calvett (39). If the extraction is 

 made with boiling water, the reduced coenzyme is oxidized by air. 

 Therefore no loss occurs if some of the subsequent steps of the 

 preparation are carried out in acid medium. 



In most tissues somewhat more of the oxidized than of the reduced 

 form of cozymase was found, but in the Jensen rat sarcoma, Euler 

 and his co-workers found a large excess of dihydrocozymase (20, 40). 

 Recent experiments confirm this finding, but apparently it is not 

 true for all cancerous tissues. An excess of dihydrocozymase was 

 repeatedly found in methyl-cholanthrene rat tumor, but not regularly 

 in benzpyrene tumors of mice and in Brown-Pearce rabbit carci- 

 noma (33). The data on human carcinomata are still too limited to 

 permit any conclusion. 



It must be remembered that all methods of determination in tis- 

 sues involve complications: incompleteness of extraction, limited 

 stability of the codehydrogenases in oxidized and reduced form at 

 high temperature, rapid changes in the equilibrium Co I <=s Co II, 

 enzymatic destruction upon disruption of the cells, and finally 

 errors in the methods of determination. The claims for accuracy 

 which are made by many publications dealing with coenzyme con- 

 tent of tissues seem far too optimistic. 



The growth-promoting properties of the codehydrogenases for 

 certain microorganisms (Hemophilus influenzae and Hemophilus 

 para-influenzae) can be used for detennining small quantities of 

 these compounds (41). Whereas the number of nicotinamide- 

 requiring microorganisms is rather high, only H. influenzae and 



