10] Preparation of cytochrome oxidase from beef heart

Abstract

This chapter describes the methods of solubilizing cytochrome oxidase from mitochondrial membranes. These procedures seek to limit bile salt treatment to a minimal period followed immediately by ammonium sulfate fractionation. These preparative methods each used the rationale of a “redgreen split” (sequential fragmentation) to remove reductases (complexes I, I–III, II, II–III, and III) prior to the solubilization of cytochrome oxidase (complex IV). Cytochrome oxidase, the terminal member of the mitochondrial electron-transport chain, is a membrane-bound, multisubunit protein containing two heme and two copper ion oxidation–reduction centers. It has become obvious that long-term interaction of the oxidase with bile salts, used for solubilization, is detrimental to overall structural and functional integrity. Unless otherwise specified, all manipulations are performed at 0° to 4°. In short, the cytochrome oxidase preparations reported in the chapter shorten the time of preparation and exposure to bile salts, yield higher redox component ratios per milligram of protein, and exhibit excellent enzymic activity for cytochrome oxidation by molecular oxygen.