IV. BIOCHEMICAL SYSTEMS 345 



this preparation, but the re-reduction of cytochrome c will not take place 

 /// vitro unless an enzyme capable of re(lucin<>; cytocln'ome c is added. Several 

 flavoproteins capable of reducing cytochrome c have been isolated.^" One 

 of these is cytochrome c reductase, which, like Warburg's yellow enzyme, 

 has the "mononucleotide" riboflavin phosphate for a prosthetic group. 



This enzj'ine, which was isolated by Haas ct al}^ in 1940 and which 

 catalyzes the transfer of hydrogen between reduced triphosphopyridine 

 nucleotide and cytochrome c, is not to be confused with either the di- 

 aphorases, which can oxidize diphosphopyridine nucleotide, or with Haas' 

 rtavoprotein from yeast, which, like the diaphorases, contains riboflavin 

 dinucleotide as the prosthetic group. 



a. Preparation and Properties of Cytochrome C Reductase 



A specially prepared dried brewer's yeast^^ was extracted in 20° water 

 for 33 hours. After centrifuging, the resulting supernatant solution was 

 51 % saturated with ammonium sulfate at pH 4.5. The resulting precipitate 

 was suspended in 31 % ammonium sulfate, in which the enzyme is soluble; 

 again precipitated with 51 % ammonium sulfate; freed of salt by dialysis; 

 precipitated with ethanol; adsorbed on aluminum hydroxide gel and eluted 

 with alkahne ammonium sulfate ; adsorbed on tricalcium phosphate gel and 

 eluted with pH 6.1 phosphate buffer; and finally adsorbed on aluminum 

 hydroxide gel and eluted with alkaline ammonium sulfate, from which solu- 

 tion it was precipitated with 70% saturated ammonium sulfate. The cyto- 

 chrome c reductase so obtained in 1940 was considered to be 87 % pure. It 

 was estimated that the dried yeast contained 0.6 g. of this flavoprotein per 

 kilogram. 



The first preparations of cytochrome c reductase were very unstable in 

 environments not considered harmful to other flavoproteins, which may 

 explain why it was not isolated at an earlier date. The enzyme lost 30 % of 

 its activity in 2 days at 0° and would have been completely destroyed 

 after 24 hours in 33 % acetone, a condition under which Warburg and 

 Christian prepared their old yellow enzyme. Its molecular weight is about 

 75,000. Its prosthetic group, riboflavin-5-phosphate, can be interchanged^^ 

 with that of the old yellow enzyme. This fact placed this coenzyme in a 

 more significant position as a biologically important compound, since prior 

 to the isolation of cytochrome c reductase it was suspected that riboflavin- 

 5-phosphate might be an artifact. 



In 1942, Haas et al}- improved the procedure of isolation and obtained 



^^ J. B. Sumner and G. F. Somers, Chemistry and Methods of Enzymes, p. 271. 



Academic Press, New York, 1947. 

 ^' E. Haas, B. L. Horecker, and T. R. Ilogness, ./. Biol. Chem. 136. 747 (1940). 

 " E. Haas, C. J. Harrer, and T. R. Hogness, ./. Biol. Chem. 143, 341 (1942). 



