Isolation and Functional Reconstitution of Rat Liver Cytochrome Oxidase

Cytochrome oxidase is the terminal component of the respiratory chain of mitochondria. It functions as an electron carrier, between cytochrome c and oxygen, and as a proton pump, thus participating as a redox pump in the conversion of metabolic energy during the synthesis of ATP (Azzi 1980; Wainio 1983). Cytochrome oxidase has been purified from mitochondria of different sources. The most common technique used to purify beef heart cytochrome oxidase is based on the solubilization of mitochondria by cholate, followed by salt fractionation to separate the different cytochromes (Hartzell et al. 1978). Other techniques are based on hydrophobic chromatography (Nagasawa et al. 1979), and affinity chromatography (Bill et al. 1982; Rascati and Parsen 1979). An additional experimental approach, which has been often used to purify rat liver cytochrome oxidase, is based on differential solubilization using nonionic detergents of the Triton series (Ades and Cascarano 1977). Triton X-114 solubilize the b—c1 complex but not the cytochrome oxidase which can be subsequently solubilized using Triton X-l00 (Slinde and Flatmark 1976).