Studies on Problems of Cytochrome c Oxidase Assay 261 



If all of the above assumptions are correct, a given concentration of cyto- 

 chromes a plus ^3 (which seem to occur in a constant ratio in mammalian 

 tissues) should represent a definite amount of cytochrome c oxidase activity. 

 In a purified preparation of cytochromes a plus a^ this has been shown to be 

 so (Smith, 1955). With this type of preparation the dilution of the enzyme in 

 the assay system must be great enough to eliminate the inhibitory effect of the 

 cholate in the preparation on the enzyme activity. As discussed below, the 

 activity of the oxidase in the purified preparation can be very high under 

 special conditions. On the other hand, when the oxidase activity of tissue 

 homogenates or particulate preparations from cells is assayed by variations of 

 either method (1) or (2) above, the activity often appears to be quite low com- 

 pared to the content of cytochromes a plus ^3. 



Our studies of the apparently low cytochrome c oxidase activity of many 

 cellular extracts cover several aspects of the problem: (1) It has been shown 

 that soluble cytochrome c actually inhibits cytochrome c oxidase activity 

 (Smith and Conrad, 1956). This work has been pubhshed and will be only 

 briefly summarized. (2) Other basic proteins besides cytochrome c have been 

 found to be inhibitory to cytochrome c oxidase activity, and substances 

 present in some tissue homogenates are also inhibitory. (3) Studies have been 

 made of the activities of fractions separated from homogenates of different 

 kinds in an attempt to determine which type of preparation gives maximal 

 activity. 



Although many methods have been devised for measuring cytochrome c 

 oxidase activity by varying the type of substance oxidized by the enzyme 

 system or the conditions of the assay, few observations have been made on the 

 effect of the state of the cellular extract. It should be recalled that difficulties 

 are met as a consequence of the attachment of the oxidase to insoluble 

 particulate matter within the cell and that even in purified preparations one is 

 not dealing with a water-soluble enzyme. Our studies have attempted to 

 evaluate the different methods for measuring cytochrome c oxidase activity 

 and particularly the relationship of the oxidase activity to the type of cellular 

 homogenate or fraction. 



METHODS 



Preparations 



The purified preparation of cytochromes a plus ^3 has been described 

 (Smith, 1955). 



The preparation of heart muscle particles was made by a modification of 

 the method of Keilin and Hartree (Chance, 1952). 



Mitochondria were isolated from liver (Lardy and Wellman, 1952), 

 kidney (Hollunger, 1955) and heart (Cleland and Slater, 1953). Cytochrome 

 c was removed from liver mitochondria by washing with saline (Estabrook, 

 1958). Homogenates of rat organs (10% or 20%) were prepared by grinding 



