Studies on Problems of Cytochrome c Oxidase Assay 



111 



in the determined K,n as low salt concentrations are approached. This lower value 

 for the K,n for cytochrome c, that is 3 x 10"' m, when related to the concentration of 

 endogenous cytochrome c which is normally present in such a heart muscle prepar- 

 ation, shows that exogenous cytochrome c is nearly as effective as endogenous cyto- 

 chrome c thus negating the necessity of assessing the previously low activity values 



0060- 



1^ 



o 0O40 





 -0010 



Ol 23456789 



Time, mjn 



A 



0-06 12 018 



Phosphate buffer concentration, M/l 



B 



Fig. 2. 



to the requirement for a lipocytochrome c. Other studies have shown that the in- 

 hibitory action of cations is dependent not only on their concentration but on their 

 valency charge. That is, the divalent cations, Ca++, Ba++, Mg+''", are much more 

 inhibitory than the monovalent cations, Na+, K+, and Li+. In turn, the trivalent 

 cation A1+++ is much more inhibitory than the divalent cations. 



By following spectroscopically the change in steady-state reduction of the exogenous 

 cytochrome c present during succinate oxidation by such a system, it is possible to 

 assess the locus of inhibition as between reduced cytochrome c and cytochrome 

 oxidase. The decrease in oxygen uptake accompanied by the increase in steady-state 

 reduction of the exogenous cytochrome c as the cation concentration increases is 

 summarized in Figs. 2a and 2b. 



The inhibitory effect of cations as determined from the change in K^ of the system 

 for exogenous cytochrome c is also largely dependent on the enzyme concentration. 



