Studies on Problems of Cytochrome c Oxidase Assay 273 



acts to block interactions physically. The relatively small inhibitory effect of 

 high concentrations of cytochrome c on the oxygen uptake of heart muscle 

 particles indicates that the masking of the oxidase by cytochrome c inhibits 

 the oxidative reaction of the oxidase with cytochrome c in solution, but may 

 not block the reaction between the oxidase and the endogenous cytochrome c 

 of the particles. 



Preliminary observations on the effect of salmine on the succinate-cyto- 

 chrome c reductase activity of particulate preparations indicate that in this 

 case also the salmine has an inhibitory effect. The postulate might be made 

 that the surface of the particles bearing the electron transport chain has 

 structural parts that can bind basic proteins such as salmine strongly. This 

 binding prevents the interaction of the particulate enzymes with cytochrome c 

 in solution. The poor accessibility of soluble cytochrome c to a catalytic site 

 on heart muscle particles has been suggested by Keilin and Hartree (1955). 



Thus there appear to be at least two structural effects which can inhibit 

 the reaction of the particulate oxidase with cytochrome c in solution. Some 

 structural barrier inhibits the reaction with cytochrome c of the oxidase of in- 

 tact mitochondria and of some kinds of particulate preparations derived from 

 the breakdown of the mitochondria. With the latter, low concentrations of 

 surface active agents such as cholate or deoxycholate can remove the 

 inhibition (Smith and Stotz, 1954; Mackler and Green, 1956). The present 

 data show that the oxidase can also be masked by binding of some proteins. 

 To what extent the two effects which mask the atjility of the oxidase to react 

 with soluble cytochrome c are interrelated is not yet clear. But the effects are 

 such that different maximal oxidase activities (expressed in terms of content of 

 cytochromes a + flg) are observed with preparations from different rat organs, 

 showing that the extent of the structural inhibitory effects varies in different 

 tissues. 



Although observations on the factors which may affect the oxidation of 

 soluble cytochrome c by cytochrome c oxidase of tissue homogenates on 

 particles indicate many difficulties in assessing variations in apparent 

 oxidase activity, further experiments of this kind may lead to some insight into 

 the nature of the reaction site of the enzyme. 



Gamble (1957) has shown that liver mitochondria or mitochondrial frag- 

 ments suspended in media of low ionic strength can definitely bind cytochrome 

 c or salmine and that under these conditions aggregation of the mitochondria 

 or fragments is observed. The binding of cytochrome he describes appears to 

 be irrelevant to the present experiments, since it was not observed in solutions 

 of phosphate buffer comparable to those used in the experiments reported 

 here. 



SUMMARY 

 Measurements of cytochrome c oxidase activity have been made by obtaining 

 the first order velocity constant for the oxidation of ferrocytochrome c 



