344 RIBOFLAVIN 



The general diaphorase reaction for the oxidation of either the di- or 

 triphosphopyridine nucleotide is as follows: 



H2-DPN + diaphorase — > DPN + reduced diaphorase 



Reduced diaphorase + methylene blue 



— » Diaphorase + leucomethylene blue 



Leucomethylene blue + 62—^ Methylene blue + H2O2 



Abraham and Adler^^ have indicated that there are two separate di- 

 aphorases, one for reduced coenzyme I and one for reduced coenzyme II. 



5. Straub Flavoprotein, Soluble Diaphorase 



The chemical nature of the diaphorases was clarified when Straub*^ • *^ 

 succeeded in separating a soluble flavoprotein from pig heart. This prepa- 

 ration had all the catalytic properties of diaphorase, and it was concluded 

 that they were the same compound in different physical states. The essen- 

 tial step of the preparation was the heating of the crude diaphorase prepa- 

 ration at pH 4.6 in 2 % ammonium sulfate and 3 % ethyl alcohol at 43° for 

 10 to 15 minutes. Some 14 % of the enzyme activity went into solution and 

 could be centrifuged from the insoluble particles. 



Straub's flavoprotein is much more stable to heat than diaphorases which 

 are attached to insoluble particles. The soluble flavoprotein from the pig 

 heart can be heated to 80° without destroying its catalytic activity. Boihng 

 the solution liberates the prosthetic group, flavin adenine dinucleotide. 



According to Corran et al.,'^^ the oxidation of both reduced coenzyme I 

 and reduced coenzyme II can be catalyzed by this flavoprotein. Abraham 

 and Adler^^ showed that the heart flavoprotein had very little activity 

 against reduced coenzyme II, and it is now customary to catalog the oxi- 

 dation of reduced diphosphopyridine nucleotides as a specific function of 

 soluble diaphorase. 



The catalytic effect of heart flavoprotein on oxygen uptake in lactic 

 systems and in maUc, tissue phosphoric, and a-glycerophosphoric enzyme 

 systems has been stressed by Corran et al.^^ The turnover number of this 

 enzyme was found to be 8500. 



6. Cytochrome C Reductase of Yeast 



In the living cell cytochrome c is reversibly reduced and oxidized. Cyto- 

 chrome oxidase provides a mechanism for the participation of oxygen in 



<6 E. P. Abraham and E. Adler, Biochem. J. 34, 119 (1940). 



" F. B. Straub, Nature 143, 76 (1939). 



« F. B. Straub, Biochem. J. 33, 787 (1939). 



49 H. S. Corran, D. E. Green, and F. B. Straub, Biochem. J. 33, 793 (1939). 



