482 NIACIN 



might be a hexose'* and the manner of linkage of the various molecules 

 was not clarified. 



Warburg and Christian encountered their "Co-ferment" as a factor 

 necessary for the action of Zwischenferment, an enzyme which oxidized 

 glucose 6-phosphate.^ They isolated the coenzyme in a "pure" form from 

 hemolyzed horse erythrocytes by a rather involved procedure employing 

 acetone precipitation of the red cell stromata, fractionation with mercuric 

 acetate and barium hydroxide, precipitation from methanol-HCl with ethyl- 

 acetate, and further fractional precipitation with lead acetate and alcohol. 

 A fluorescent contamination which interfered with spectrophotometric 

 studies could be removed by bromine treatment.** 



2. DPN was the first coenzyme to be discovered and the second found 

 to have nicotinamide as a part of its molecule. It was found by Harden 

 and Young^ as a material which could be dialyzed from the complex zymase 

 system of yeast, the system which catalyzes the alcoholic fermentation of 

 carbohydrates. Actually, this soluble dialyzable component of the zymase 

 system contained, as later shown, two coenzymes, cozymase (DPN) and 

 phosphorylating coenzyme (adenosine phosphate). 



By 1933 it was known, from the work of von Euler and Myrback,!" that 

 cozymase was an adenine nucleotide. About a year after Warburg and 

 Christian identified nicotinamide as part of the TPN molecule, von Euler 

 et al.^ isolated a base from cozymase which they believed to be nicotinamide. 

 Warburg and Christian^ also isolated a pyridine base from cozymase and 

 published convincing evidence of its identity as nicotinamide. From the 

 work of both groups, it was evident that cozymase was closely related to 

 TPN, since it contained the same structural elements differing only in that 

 it contained one less phosphate group. 



DPN was originally isolated from brewer's or baker's yeast, although 

 animal tissues were used in much of the early work. The first "pure" 

 preparations were obtained from yeast^' *^ by a procedure which consisted 

 of hot water extraction, lead acetate precipitation of impurities, coenzyme 

 precipitation in consecutive steps as the phosphotungstate, Hg, Ag, and 

 Cu salts, followed by removal of contaminations by Ba and Pb treatment, 

 alcohol fractionation, and purification with aluminum oxide. In using ani- 

 mal tissue (erythrocytes) as a source material, Warburg and Christian^ 

 were able to isolate not only DPN but TPN and adenosine phosphate as 

 well. They separated the three nucleotides, utilizing the fact that the 

 barium salt of adenosine phosphate is only slightly soluble in water. The 

 barium salt of TPN is only slightly soluble in dilute alcohol, whereas the 



8 O. Warburg and W. Christian, Biochem. Z. 242, 206 (1931). 



8 A. Harden and W. J. Young, J. Phi/sioJ. 32 Proc. 1904 (1905); Proc. Roij. Soc. 



(London) B77, 405 (1906). 

 10 K. Myrback, Ergeb. Emymforsch. II, 139 (1933). 

 '1 H. von Euler and F. Schlenk, Svensk Kern. Tidskr. 48, 135 (1936). 



