Studies on Problems of Cytochrome c Oxidase Assay 267 



The inhibitory effect with increasing cytochrome c concentrations on the 

 purified oxidase is greater than with the enzyme attached to tiie heart muscle 

 particles. This could mean that the oxidase of the purified preparation is 

 more exposed to form the unreactive binding with cytochrome c than is the 

 enzyme which is still a part of the respiratory chain particles. 



As far as the methodology of cytochrome c oxidase assay is concerned, the 

 data indicate that: 



(1) If comparative studies of the oxidase activity of a given kind of prepara- 

 tion are to be made, the concentration of cytochrome c must be held constant 

 throughout the tests. Then only comparative values will be obtained, since 

 the inhibitory effect of the cytochrome c will be present. If oxidase activities 

 of different tissues or different kinds of preparations are to be studied, the 

 inhibitory effect of cytochrome c on each kind of preparation must also be 

 compared. 



(2) The high concentrations of cytochrome c usually employed in the 

 manometric method and in most colorimetric methods of cytochrome c 

 oxidase assay will result in a greatly inhibited enzyme. 



When the velocity constant of the reaction of the purified oxidase is 

 measured at very low concentrations of cytochrome (around 1 /^m), a very 

 active enzyme is apparent. If a second order velocity constant is calculated by 

 dividing the observed first order constant by the concentration of cytochrome 

 ^3, values as high as 10^ m~^ sec~^ are obtained at 25°C (Smith and Conrad, 

 1956). 



Ejfect of Proteins Other than Cytochrome c on Cytochrome c Oxidase Activity 



Since cytochrome c is a protein with an isoelectric point above pH 10, the 

 effect of other basic proteins on the cytochrome c oxidase activity was tested. 

 Table 1 shows the effect of different concentrations of salmine on the oxidase 

 activity of heart muscle particles, all other components being kept constant in 

 the test. The concentration of cytochrome c in the mixture was 7 /tM. Figure 

 5 plots the oxidase activity with increasing concentrations of cytochrome c in 

 the presence of a constant salmine concentration. The data show that at low 

 concentrations salmine has a greater inhibitory effect than does cytochrome c. 

 The inhibitory effect of cytochrome c is almost entirely ehminated in a 

 preparation inhibited by salmine. Apparently salmine binds at or near the 

 same site as does the cytochrome c. 



With these effects of basic proteins on the oxidase in mind, we were led 

 into a comparison of the cytochrome c oxidase activity of homogenates of 

 some animal tissues with their content of cytochromes a and O3. This 

 examined the possibility that the low oxidase activity sometimes observed in 

 homogenates results from inhibition of the oxidase activity by substances 

 present in the homogenates. Table 2 shows some results obtained with rat 

 liver homogenates. The oxidase activity of washed particles is about 4-5-fold 



H.E. — VOL. I — T 



