STUDIES ON PROBLEMS OF CYTOCHROME c 

 OXIDASE ASSAY 



By LuciLE Smith and Helen Conrad 



Department of Biochemistry, Dartmouth Medical School and 

 Johnson Research Foundation, University of Pennsylvania 



INTRODUCTION 



The best evidence available indicates that the enzyme cytochrome c oxidase 

 is a combination of cytochromes a and a^, and that the cytochrome % reacts 

 directly with oxygen (Keilin and Hartree, 1938, 1939; Chance, 1953; Smith, 

 1955). The combination can rapidly oxidize ferrocytochrome c, either when 

 the cytochromes a, a^ and c are all attached to insoluble particulate material 

 or when the cytochrome c is in solution and the cytochromes a and a^ are 

 particle-bound. The reaction is usually represented as: 



cytochrome c -^ cytochrome a -^ cytochrome a.^ -^ O2 



The affinity of cytochrome a^ for oxygen is very high (Ludwig and Kuby, 

 1955; Chance and Williams, 1955). 



There are several ways of assaying for cytochrome c oxidase: (1) The rate 

 of oxidation of soluble ferrocytochrome c can be measured spectrophoto- 

 metrically, the oxygen in solution being in ample excess. (2) The rate of 

 oxygen uptake can be measured in the presence of a substance which will 

 continuously reduce the cytochrome c nonenzymically; alternatively the 

 rate of oxidation of the reducing agent, which may be a dye which changes 

 colour on oxidation, is measured. 



reducing agent -^ cytochrome c -^ cytochrome a -> cytochrome % -^ O2 



Here the assumption is made that the rate of reduction of the cytochrome c is 

 rapid compared to the oxidation of the ferrocytochrome c by the oxidase. 

 This assumption has been shown in one instance to be incorrect (Conrad, 

 1951). (3) A direct method of assay would be to measure spectrophoto- 

 metrically the content of cytochromes a and a^ in the preparation. The 

 extinction coefficients for the difference between reduced and oxidized cyto- 

 chrome ^3 and the extinction coefficient for the carbon monoxide compound 

 of cytochrome a^ have been measured (Chance, 1953). In turbid preparations 

 such as tissue homogenates or mitochondria this type of measurement is 

 difficult, except with specialized apparatus (Chance, 1954). 



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