Studies on Problems of Cytochrome c Oxidase Assay 



269 



oxidase activity of homogenates. Although we have not carried out experi- 

 ments with plant tissues, some published data show that in homogenates of 

 some plant tissues the oxidase activity is low or absent, but appears on 



20 



Cytochrome C cone • 



40 

 // M 



Fig. 5. The effect of cytochrome c concentration on the velocity constant of the 



oxidase reaction in the presence and absence of salmine. The oxidase preparation 



was a suspension of rat heart sarcosomes treated with 100 vol of distilled water. 



The concentration of salmine in the test was 1 //m. 



preparation of washed particles, indicating that something inhibitory has 

 been washed away (James, 1956; Simon, 1957). 



Studies of Oxidase Activity of Fractions of Tissue Homogenates 



Some experiments were carried out which attempted to give some idea 

 about the possible localization of the substances in homogenates which are 

 inhibitory to cytochrome c oxidase. The oxidase is a part of the mitochondria, 

 but the oxidase of intact mitochondria suspended in isotonic sucrose does not 

 rapidly oxidize soluble ferrocytochrome c in the suspending medium. The 

 oxidase does react rapidly after the mitochondria have been exposed to water 

 or dilute buffer solutions. We have investigated the oxidase activity of mito- 

 chondria to determine under what conditions maximal oxidase activity can 

 be obtained. When a small volume (5-10 /d.) of a concentrated mitochondrial 

 suspension in isotonic or hypertonic sucrose is added to ferrocytochrome c in 

 0-05 M phosphate buffer, the changes in optical density during the first 20 sec 

 are not entirely due to the oxidation of cytochrome c, since the usual straight 

 lines on a semilogarithmic plot are not obtained. These early changes in 

 optical density, which presumably result from changes in light scattering as 

 the mitochondria swell (Claude, 1946), are not significant after about 20 sec. 

 However, it was found that if the mitochondria were allowed to stand diluted 



