166 A SYMPOSIUM ON RESPIRATORY ENZYMES 



The reduction of cytochrome c by hydroquinone is very rapid; 

 hence the rate-controHing reaction here is the oxidation of the re- 

 duced cytochrome. Several curves such as those in Figure 3 have 

 been found to comply with the laws of a typical enzyme-substrate 

 complex (75), indicating that the oxidase and cytochrome c form 

 such a complex. A study of such curves in relation to the effect of 

 cyanide and carbon monoxide led to the conclusion that the action 

 of these inhibitors on the reaction was concerned with the oxidase 

 component (75). 



On the other hand, in the presence of an excess of cytochrome 

 c the velocity of oxidation of hydroquinone (correcting for autoxida- 

 tion) was directly proportional to the amount of oxidase added. This 

 offers a method of estimating, in arbitrary units, the cytochrome 

 oxidase activity of tissues. 



From hydroquinone and p-phenylenediamine tests it appears that 

 successive acetic acid precipitations remove the larger part of the 

 cytochrome c and some of the cytochrome h. By two precipitations 

 with acetic acid and a long dialysis an active oxidase preparation 

 can be obtained which shows very little cytochrome c or h, al- 

 though the oxidase activity is likewise greatly diminished. 



Determination and Distribution of Cytochrome c and 

 Cytochrome Oxidase 



Junowicz-Kochalaty and Hogness (76) have developed a method 

 for estimating cytochrome c in tissues. Relatively large amounts 

 (100 grams of tissue) are worked up through the initial steps of 

 Keilin's isolation procedure (5) to the point where traces of hemo- 

 globin and myoglobin are the principal colored impurities. The 

 cytochrome c is then measured spectrophotometrically. The use of 

 measurements at three wave lengths permits of calculations to cor- 

 rect for the hemoglobin and myoglobin. These authors found pigeon 

 breast muscle and beef heart muscle high in cytochrome c, tumor 

 tissue very low. 



Stotz (77) has developed a method for determining cytochrome c 

 in rat tissues. The ground tissue is extracted with trichloracetic acid, 

 the extract neutralized to eliminate further inactive protein, and the 

 cytochrome precipitated by phosphotungstic acid. After solution in 

 dilute ammonia, the phosphotungstate is eliminated with barium. 

 The final solutions are tested manometrically for their power to 

 accelerate oxidation of hydroquinone by a heart muscle oxidase prep- 

 aration. A calibration curve must be prepared, pure cytochrome c 

 being used. 



