NICOTINAMIDE NUCLEOTIDE ENZYMES 109 



(8). Filtration through a column of activated alumina has also been 

 employed successfully (15). 



Recently some modifications in the preparation of cozymase as 

 given in Table 3 have been described (10-13), The improvements 

 consist mainly in omitting some of the purification steps, which can 

 be done without complications if a good quality of yeast is used 

 as the source material. B. J. Jandorf has introduced the adsorption 

 of cozymase on charcoal in his method of preparation (13). S. Ochoa 

 has developed a method in which muscle tissue is used as source 

 material (11), and P. Ohlmeyer has described a method for the 

 preparation of dihydrocozymase (10). From 10 kilograms of yeast 

 one gram of almost pure, or 0.5 gram of pure codehydrogenase I is 

 obtained. The yield of codehydrogenase II from 1000 liters of 

 erythrocytes is about 2.5 grams of almost pure, or 1.0 gram of abso- 

 lutely pure preparation. The coenzymes precipitated from aqueous 

 solution by organic solvents are not crystalline. Table 4 shows the 

 composition and properties of codehydrogenase I and II. 



The most important part of the work on the structure of the co- 

 enzymes has been concerned with the nicotinamide moiety, its mode 

 of action, and the linkage between nicotinamide and the rest of 

 the molecule. This work was begun by Warburg (8) and continued 

 by Karrer and his co-workers (23a-f). Warburg showed first that in 

 codehydrogenase II the nicotinamide reacts with two atoms of 

 hydrogen in the presence of substrate and apoenzyme, forming a 

 dihydro compound. This compound can also be obtained by reduc- 

 tion with hydrosulfite in a shghtly alkaline medium. The reduced 

 coenzyme has an absorption maximum at 340 n\\i, whereas the maxi- 

 mum at 260 miJ, has lost some of its intensity by the reduction (see 

 Figure 1). By reoxidation the original state is restored. 



Catalytic hydrogenation yields an uptake of six hydrogen atoms 

 by the oxidized coenzymes and of four hydrogen atoms by the 

 biologically reduced coenzymes. Experiments with adenine and its 

 derivatives showed that under the same conditions these compounds 

 are very slowly reduced by catalytic hydrogenation, whereas free 

 nicotinamide exhibits the same properties as the coenzymes upon 

 catalytic reduction (5). It should be remembered that the catalytic 

 reduction which leads to the hexahydro compounds is irreversible, 

 and the products obtained are inactive as coenzymes. These ex- 

 periments demonstrated that the place of the reversible (biological) 

 reduction— i.e., the center of the coenzyme activity— is the nicotin- 

 amide nucleus. 



