Electioiuelric and Other Studies of Cytochrome c 



373 



The copper catalysis of ascorbate oxidation is well known (see, for example, 

 Barron, de Meio and Klemperer, 1935) and up to about 10^^ g atom/ml the 

 rate of oxidation is directly proportional to the concentration of copper. It 

 seemed hkely that the '0-34 % Fe' content samples were inhibiting an oxidation 

 of ascorbate catalysed by traces of copper present as an impurity in the 

 reagents. A series was therefore run in which there was a constant added 



0.5 10 



Cytochrome c cone, 



Fig. 2. Influence of cytochrome c fractions on ascorbate oxidation. (Determina- 

 tions using Warburg manometers, 007 m sodium phosphate buffer, pH 7-3 and 

 0-02 M sodium ascorbate, at 37°C.) 



I. With added copper (I /tig J flask). 



Curve A: Preparations: (i) before resin column treatment, and (ii) colourless 

 protein fraction separated electrophoretically. 



Curve B: Preparations: (iii) after resin column treatment, and (iv) after electro- 

 phoresis, and (v) after treatment with TCA. 

 Curve C: (vi) cytochrome fraction separated on resin column. 



II. No copper added. 



Fractions (i) to (v) inclusive and also 10~^ m EDTA lowered the oxygen uptake to 

 the bottom X; fraction (vi) catalysed the oxygen-uptake to the top X. 



concentration of copper and varying amounts of the different cytochrome c 

 fractions (Fig. 2). Three distinct types of curves were obtained. The '0-34% 

 Fe' cytochrome c gave a value the same as in the absence of added copper 

 until the point corresponding to approximately 4 cytochrome c molecules/ 

 copper atom, after which there was a rapid rise in uptake inversely pro- 

 portional to the cytochrome concentration. The '0-43% Fe' cytochrome c 

 inhibited the ascorbate oxidation to a much lesser extent than the above 

 material. This indicated that combination was in the main occurring between 

 the copper and a colourless protein fraction which could be removed electro- 

 phoretically (Theorell and Akeson, 1941) or chromatographically (Paleus 



