458 NIACIN 



7. Isolation of Coenzymes I and II 

 a. Coenzyme I (Diphosphopyridine nucleotide, DPN, cozymase) 



This substance has not yet been isolated in an absolute chemically pure 

 form/^'* It can, however, be prepared in 95 % or better purity. Ohlmeyer^^ 

 obtained a product of exceptional purity, as judged by extinction coefficients 

 at 340 mju and other criteria, by isolating reduced DPN from a purified 

 oxidized DPN preparation (see p. 483). The early investigations of von 

 Euler and his group and Warburg and associates in isolating this coenzyme 

 have been cited elsewhere (p. 480 and 482). The methods applied by these 

 workers to purify DPN and to separate DPN from TPN and other impuri- 

 ties have been outlined in Section IV. It should be noted that none of 

 the procedures described to date for isolating "pure" DPN (or TPN) has 

 yielded crystalline products. 



Improved and less cumbersome procedures have been devised in more 

 recent years which permit the isolation of DPN in a purity quite satis- 

 factory for most biochemical work. Wilhamson and Green^^ and Sumner 

 ei, al}"^ have devised methods which have found considerable use. These 

 and other methods have been reviewed by LePage.^^ Still more recently 

 Kornberg and Pricer*^ have described a modification of Williamson and 

 Green's method which yields DPN in about 95 % purity. This method is 

 attaining increasingly wide acceptance and use. The coenzyme is extracted 

 from starch-free baker's yeast with hot water. This extract is treated with 

 basic lead acetate, and DPN precipitated with silver nitrate. The precipi- 

 tate is decomposed with H2S and the DPN again precipitated with acetone. 

 The product at this stage has a purity of 76 to 83 %. Further purification 

 is achieved by adsorption on Dowex-1 resin, elution with 0.1 A'' formic acid, 

 and reprecipitation with acetone. Other investigators have also employed 

 ion exchange chromatography to purify DPN.^° 



6. Coenzyme II (Triphosphopyridine nucleotide, TPN) 



TPN has been isolated in a form almost as pure as DPN, but it has not 

 been completely purified. Warburg et al.^' ^ were the first to describe a 

 method for isolating purified TPN. The details of their procedure are de- 



44a Jy Wallenfels and W. Christian [Angew. Chem. 64, 419 (1952)] have recently an- 

 nounced the crystallization of DPN as a quinine salt. This product crystallized as 

 fine, almost featherlike needles arranged in sheaves and gave a melting point be- 

 tween 162 and 170° with decomposition. 



" P. Ohlmeyer, Biochem. Z. 297, 66 (1938). 



^6 S. Williamson and D. E. Green, J. Biol. Chem. 135, 347 (1940). 



^^ J. B. Sumner, P. S. Krishnan, and E. B. Sisler, Arch. Biochem. 12, 19 (1947). 



"8 G. A. LePage, Biochem. Preparations 1, 28 (1949). 



*^ A. Kornberg and W. E. Pricer, Jr., Biochem. Preparations 2, (1952). 



5» J. B. Neilands and A. Akeson, J. Biol. Chem. 188, 307 (1951). 



