iv. biochemical systems 343 



3. Haas Enzyme 



III n»3S, .shortly aft(M- Warburg- and Cliri.stian'''' -■^' " had idoiitifiod flavin 

 adenine dinueleotide as the proslhetic group of D-amiiio acid oxidase, Ilaas''* 

 isolatetl a flavoprotein from bottom yeast which contained isoalloxazine 

 adenine dinueleotide as the prosthetic group. The catalytic properties 

 were qualitati\'ely the same as tliose of the old yellow enzyme. Haas' en- 

 zyme was somewhat more rapidly reduced by reduced triphosphopyridine 

 luicleotide, but the rate achieved was still too slow to be of biological im- 

 portance. At physiological tensions of oxygen this flavoprotein was virtually 

 non-oxidizable, and in reconstructed systems it required an intermediate 

 carrier, like methylene blue, for its reaction with molecular oxygen. 



The Haas enzyme contained about 0.7 % flavin adenine dinueleotide. 

 The molecular weight was estimated as 70,000. The specific protein could 

 not form a catalytically active complex with riboflavin phosphate. 



4. DiAPHORASE 



The term diaphorase (from the Greek ^ta^epeti/ = transfer) is applied 

 to a group of flavoproteins capable of catalyzing the oxidation of reduced 

 pyridine nucleotides. Diaphorase, also called coenzyme factor, was dis- 

 covered independently by von Euler and Hellstrom^^ and by Dewan and 

 Green.'*" • •*! It has since been found in bacteria, yeast,^^ plants,'*^ blood,'*^ 

 milk,*^ animal muscle,^^- ^'' ''^ brain, kidney, intestine, thyroid, and pla- 

 centa. 



Preparations of diaphorase are essentially alkaline phosphate extracts of 

 ground tissues. These crude preparations are more active" per milligram 

 of dried weight than Warburg's pure old yellow enzyme. Most diaphorases 

 are associated with insoluble particles which complicate the task of puri- 

 fication. 



The prosthetic group in diaphorase is isoalloxazine adenine dinueleotide. 

 Heating to 60° does not destroy the enzymatic activity. Solutions of di- 

 aphorase are yellow and show a green fluorescence. In this respect they 

 differ from other flavoproteins which do not fluoresce. The addition of 

 sodium dithionate or reduced diphosphopyridine nucleotide decolorizes the 

 enzyme solution. 



" O. Warburg and W. Christian, Biochem. Z. 296, 294 (1938). 



'« E. Haas, Biochem. Z. 298, 378 (1938). 



" H. von Euler and H. Hellstrom, Z. physiol. Chem. 252, 31 (1938). 



^o J. G. Ucwan and D. E. Green, Nature 140, 1097 (1937). 



" J. G. Dewan and D. E. Green, Biochem. J. 32, 626 (1938). 



« D. E. Green and J. G. Dewan, Biochem. J. 32, 1200 (1938). 



" H. S. Corran and D. E. Green, Biochem. J. 32, 2331 (1938). 



'* H. von Euler and G. Gunther, Z. physiol. Chem. 256, 229 (1938). 



" H. von Euler and K. Ilaase, Naturwissenschaften 26, 187 (1938). 



