THE FLAVOPROTEINS 145 



Then the cell is filled with buffer, and when all components of the 

 reducing system except the reductase are placed before the filament, no 

 bands appear. However, when the reductase is added, the two visible 

 bands of cytochrome c gradually appear as intense broad black lines. 

 The cytochrome is then reduced. 



To oxidize the cytochrome c, H2O2 is added. No reaction occurs, i.e., 

 the black bands do not disappear, until the enzyme cytochrome c peroxi- 

 dase is added. Upon the addition of this latter enzyme, the two bands 

 disappear and reappear again as soon as the HgO, is completely reduced. 



Not only can this system be used to determine the concentration 

 of cytochrome c reductase, but it can serve as an analytical method 

 for the determination of any one of the constituents. In fact, my two 

 collaborators, Haas and Harrer (9), have only recently worked out 

 the conditions necessary for a relatively simple determination for 

 triphosphopyridine nucleotide. By using glucose and adenosine 

 triphosphate instead of hexose monophosphate, this system should 

 serve as a method for determining the relative concentrations of 

 either mutase, the enzyme that converts glucose-1-phosphate to 

 glucose-6-phosphate, or hexokinase, the enzyme that directly phos- 

 phorylates glucose-6-phosphate. 



Haas and Harrer (9) have also made very accurate measurements 

 on the rate of oxidation of the reductase by cytochrome c and the 

 rate of reduction by dihydrotriphosphopyridine nucleotide under 

 a great variety of conditions, and as a result we have demonstrated 

 by these kinetic measurements that the reductase forms com- 

 plexes with both the dihydrotriphosphopyridine nucleotide and with 

 the cytochrome c. The approximate first-order reaction velocities 

 have been determined, as have also the heat of dissociation of the 

 complexes and the energy of activation of the first-order reactions. 

 The tentative results of these studies are embodied in Table 4. The 

 values given in the table are approximations. Although the data 



Table 4.— Properties of cytochrome c reductase 



