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LuciLE Smith and Helen Conrad 



the point marked D on curve I of Fig. 2a. When the initial rates of ferro- 

 cytochrome c oxidation are calculated (velocity constant x initial concentra- 

 tion of ferrocytochrome c) and plotted against the total cytochrome c 

 concentration, as in Figs. 3a and 3b, hyperbolic plots are obtained, similar 

 to those seen when measuring cytochrome c oxidase activity in the presence 

 of a reducing substance. The data show that the apparent 'saturating' effect 



20 40 60 



Cytochrome c cone, /^M 



Fig. 4. The effect of the concentration of cytochrome c on the velocity constant 



for the oxidase reaction of liver particles treated to remove the endogenous 



cytochrome c. In each test 10 //I. of a 20-fold dilution of the liver particles in 



0-05 M phosphate buffer was used. The temperature was 25°C. 



with increasing concentrations of cytochrome c is actually a reflection of an 

 inhibitory effect of the cytochrome c on the oxidase activity. 



The above-described experiments on oxidase preparations obtained from 

 heart muscle have been repeated with a preparation derived from rat liver 

 mitochondria treated to remove most of the endogenous cytochrome c. 

 Entirely similar plots were obtained (Fig. 4). Thus in the presence or absence 

 of endogenous cytochrome c the same kinetics are observed. 



We have interpreted our data to mean that the soluble cytochrome c 

 (either oxidized or reduced) reacts reversibly with the oxidase to form a 

 combination in which the oxidase is inhibited or in which the oxidase is so 

 masked that it cannot react with further cytochrome c in solution. Purifica- 

 tion of cytochrome c in a number of ways did not change the observed 

 kinetics, as illustrated in Fig. 2a. Thus the inhibitory effect of the cytochrome 

 c does not appear to result from an impurity in the cytochrome c solution. 



