338 



RIBOFLAVIN 



Since riboflavin is necessary for the growth of many bacteria and is a 

 common component of plant products, it is logical to assume that the 

 dinucleotide is a common constituent of the cells of most living things. 

 Yeast offers the most convenient source for preparation. Warburg and 

 Christian!^ extracted yeast at 75°; the filtrate was two-thirds saturated 

 with ammonium sulfate and extracted with phenol. The phenol extracts 

 were mixed with ether, and the dinucleotide extracted with water. The 

 ether was removed by evacuation, the aqueous solution acidified with nitric 

 acid to about pH 2, and the dinucleotide precipitated as the silver salt. The 

 precipitate was resuspended in water and decomposed with hydrogen sul- 



70 



E 20 



E 



o— 6 



2,8 



5.6 



7.0 8.4 9.8 11.2 12.6 14.0 15.4 

 7 of the active group 

 Fig. 1. Synthesis of the Warburg-Christian flavoprotein. The activity of the en- 

 zyme increases as more of the prosthetic group, riboflavin phosphate, is added to 

 form the original catalytically active flavoprotein (TheorelP"). 



fide. The dinucleotide, which was almost completely absorbed on the silver 

 sulfide precipitate, was eluted with dilute barium acetate. The eluates were 

 mixed with ammonium acetate solution and concentrated to dryness in 

 vacuo. By taking advantage of the fact that the barium salt of the dinucleo- 

 tide is twice as soluble at 60° as at room temperature, it was possible to 

 separate the barium salts of the dinucleotide from the barium salts of the 

 contaminating adenine nucleotides. 



The similarities of the dinucleotide to riboflavin phosphate in color, 

 fluorescence, and reversible reduction and oxidation have been discussed 

 (p. 336). It is less stable than riboflavin because of its tendency to hydro- 



22 p. Karrcr, P. Frei, B. H. Ringier, and H. Bendas, Helv. Chim. Acta 21, 826 (1938). 

 " O. Warburg, W. Christian, and A. Griese, Biochem. Z. 295, 261 (1938). 

 24 O. Warburg, W. Christian, and A. Griese, Biochem. Z. 297, 417 (1938). 

 26 N. H. Horowitz, J. Biol. Chem. 154, 141 (1944). 



