NICOTINAMIDE NUCLEOTIDE ENZYMES 105 



only method that has been described for preparing it in the pure 

 state. 



Codehydrogenase II occurs in much lower concentration than 

 codehydrogenase I. In Table 1 are given some typical examples 

 of the occurrence of the two coenzymes. The values are only ap- 

 proximate because crude extracts were used for the determinations. 

 With improved methods of determination it probably will be neces- 

 sary to revise these values considerably, especially those for co- 

 dehydrogenase II. 



Table 1.— Some examples of the occurrence of the codehydrogenases 



I and II 



Codehydrogenase content in micrograms 



_, . , . , per gram of fresh material 

 Material exammed 



Co I Co II 



Bottom yeast >500 <10 



Top yeast >500 5-10 



Erythrocytes (horse) 100 >12 



Liver (rat) >200 30 



Muscle (rat) 200 50 



Kidney (rat) 160 40 



It is remarkable that despite the low content of codehydrogenase 

 II in the source material and the difficulty and lengthiness of the 

 isolation procedure, the preparation of codehydrogenase II in a 

 pure state, the isolation of nicotinamide therefrom, and the demon- 

 stration of its mode of action were carried out by Warburg and 

 Christian in a remarkably short time. This work greatly facilitated 

 the isolation of nicotinamide from cozymase and the subsequent 

 work on this coenzyme in the Stockholm institute. 



In Tables 2 and 3 the methods of isolating the two compounds are 

 shown schematically. The procedures are diflFerent in the two cases, 

 but typical steps of purification known from earUer work in nucleo- 

 tide chemistry are involved in both instances. Significant steps, 

 some of them new, in the preparation of codehydrogenase II are the 

 following: removal of proteins by acetone; fractionation of the crude 

 mixture of nucleotides as barium salts, yielding adenosine polyphos- 

 phate and coenzyme I as by-products; solution of the coenzyme in 

 methanol-hydrochloric acid and reprecipitation by ethylacetate (5, 8). 



/ '-4 AT-^^-i^ ^^\ C 



