Studies on Problems of Cytochrome c Oxidase Assay 271 



in buffer for varying lengths of time at room temperature, the cytochrome c 

 oxidase activity increased somewhat. This effect is illustrated in the data of 

 Table 3 obtained with guinea pig kidney mitochondria. As found by others, 

 the oxidase activity is low when the assay is run in isotonic sucrose, as com- 

 pared with the same preparation in 0-05 M phosphate buffer, although the 

 activity of the enzyme in heart muscle particles is nearly the same in the two 

 solutions. Similar observations were made with rat heart sarcosomes; these 

 are summarized in Table 4. 



About the same maximal cytochrome c oxidase activity (expressed in terms 

 of cytochromes a and ^3) can be obtained with hypotonically treated mito- 

 chondria after standing and with washed particles isolated from a water 

 homogenate. Thus the inhibitory substances do not appear to be present in 

 the mitochondria. 



A more interesting observation is that the maximal oxidase activity that 

 could be obtained was found to be different for different tissues. Table 5 



Table 5. Cytochrome c oxidase activity of several tissues expressed 

 in terms of content of cytochromes a plus o3 



The oxidase assay and the method for measuring the difference in optical density between 



oxidized and reduced cytochromes a plus a^ are described in the text. The concentration 



of cytochrome c in the test mixture in each case was 15 fiM. 



summarizes some representative values obtained with preparations from 

 several rat organs, the assays being run at a constant cytochrome c 

 concentration. 



DISCUSSION 



Taken all together, the various data show that conclusions based upon 

 measurements of cytochrome c oxidase activity may be quite misleading. In 

 such experimentation the following observations must be kept in mind: 



(1) The oxidase activity of homogenates, washed particles or purified 

 preparations will depend upon the (total) concentration of cytochrome c in the 

 test system. Thus in a series of experiments comparing activities of a given 

 kind of preparation, the concentration of cytochrome c must be kept constant. 

 The lower the cytochrome c concentration, the higher will be the activity 

 observed, when the activity is expressed as the first order velocity constant. 



