IV. BI()(II1.MI(\T> SYSTEMS 337 



2. Riboflavin' Piu)srn.\TE 



Ban»!;a d al}'^ obtaiiuHl a yellow .sulxstance from heart muscle in 1932 

 whieii may have been the first preparation of riboflavin phosphate. How- 

 ever, the yellow enzyme which Warbnrj!; and Cliristian' obtained from 

 yeast in the same year was more thoroughly investigated. They purified an 

 aqueous extract of autolyzed bottom yeast by treatment with lead sub- 

 acetate, removed excess lead with phosphate, and precipitated the yellow 

 enzyme in the form of a viscous oil at low temperature with carbon dioxide 

 and acetone. After reprecipitation from acetone and precipitation with 

 methanol at 0°, a dry product was obtained which could be readily disso- 

 ciated into a colorless protein and a yellow prosthetic group. The latter 

 was later proved to be riboflavin phosphate. 



Theorell'^ has shown that the combination of riboflavin phosphate with 

 the apoenzyme could be reversibly dissociated as follows: When a solution 

 of the yellow enzyme was dialyzed against 0.02 .V HCl at 0°, the dialyzate 

 was slowly decolorized. The colored group (riboflavin phosphate) passed 

 through the membrane, and the colorless protein remained behind. The 

 protein was changed to a metaprotein by its contact with the dilute acid 

 (i.e., precipitated when brought to pH 7), but when the protein was di- 

 alyzed against water to remove all traces of hydrochloric acid, 50 to 70 % 

 of the metaprotein was renatured. This renatured protein was now capable 

 of recombination with the coenzyme riboflavin phosphate to produce a 

 complex with all the properties of the original yellow enzyme. 



The combination between the coenzyme and the apoenzyme^" takes 

 place in a stoichiometric manner as shown in Fig. 1. 



The reversible dissociation procedure of Theorell, which requires several 

 days for its completion, has been replaced by a simpler method by Warburg 

 and Christian'-^ in which a good yield was obtained in about 1 hour. To a 

 solution of yellow enzyme, purified by electrophoresis, an equal volume of 

 saturated ammonium sulfate was added. Sufficient 0.1 tV hydrochloric acid 

 was added to this solution at 0° to shift the pH to about 2.8. The colorless 

 precipitate formed contained 78 % of the apoenzyme in native form while 

 the coenzyme remained in the supernatant fluid. Resynthesis was accom- 

 plished by merel}^ remixing the components. 



3. Riboflavin Adenine Dinucleotide 



The dinucleotide is widely distributed in animal tissues and in micro- 

 organisms. It has been isolated from liver, kidney, muscles, tumor tissue, 

 yeast,'^' '^' ^^"-'* and Neurospora.-^ 



'» I. Banja, A. Szent-Gj'orgyi, and L. Vargha, Z. physiol. Chcm. 210, 288 (1932). 



"> H. Theorell, Biochem. Z. 275, 344 (1934). 



2« 11. Theorell, Biochem. Z. 278, 263 (1935). 



2' O. Warburg and W. Christian, Biochem. Z. 298, 368 (1938). 



