144 A SYMPOSIUM ON RESPIRATORY ENZYMES 



The cytochrome c is oxidized by oxygen through the intermediation 

 of an enzyme or enzymes known as cytochrome oxidase, and by 

 hydrogen peroxide with cytochrome c peroxidase as the catalyst 

 (31). The cytochrome c oxidase, or one component of it, is probably 

 Warburg's oxygen-carrying ferment, but it has not yet been defi- 

 nitely identified. Nor do we yet understand the whole mechanism 

 involved in the oxidation of cytochrome c. 



The test for determining the relative concentration of the cyto- 

 chrome c reductase involves all these components. If oxidized cyto- 

 chrome c is placed in a solution containing all the other components 

 and if the concentration of the hexose monophosphate is in excess, 

 the cytochrome c will be reduced, and the rate at which it is re- 

 duced will depend upon the concentrations of the other components : 

 Zwischenferment, triphosphopyridine nucleotide and the cytochrome 

 c reductase. The concentrations of all substances can be so adjusted 

 that the logarithmic rate of reduction of the cytochrome c will be pro- 

 portional to the concentration of the reductase. 



(9) -^ = K(CR) 



dt 



In this equation (CyFe+'^+) represents the concentration of the oxi- 

 dized cytochrome c and (CR) the concentration of the cytochrome 

 c reductase. The equation is an empirical one. 



It is easy to demonstrate that all components of the system must 

 be present before the cytochrome c will be reduced. In the reduced 

 state cytochrome c displays three bands with maxima at about 410, 

 520, and 550 m^., and any one of these bands may be used to deter- 

 mine the rate of reduction of the cytochrome. For most purposes the 

 band at 550 m[j. is most convenient for analytical purposes, although 

 for very dilute solutions of cytochrome c (10^ M) a wave length 

 of 418 is used. As the cytochrome c is reduced, these bands appear. 

 In the following spectroscopic demonstration, the general method 

 employed in this test is illustrated. If all components except the 

 reductase are added to the buffered solution in the absorption cell, 

 no reduction of the cytochrome c takes place until a solution of the 

 reductase is added to complete the chain of enzymatic reactions. 



Spectroscopic demonstration: The .apparatus consists of a simple 

 focusing illuminating lantern (Central Scientific Company) slightly modi- 

 fied to hold an absorption cell 2.5 cm. in diameter and 2 cm. thick. 

 A lamp with a ribbon type filament is used, and the filament, turned edge- 

 wise, is used as the slit. By placing a transmission grating in the light 

 path a spectrum is projected on the wall. 



