Biochem/physiol Actions
Cytochrome c oxidase is the principal terminal oxidase of high oxygen affinity in the aerobic metabolism of all animals, plants, yeasts and some bacteria. It is present in the mitochondria of the more highly developed cells and in the cytoplasmic membrane of bacteria. Cytochrome c oxidase catalyses the electron transfer from cytochrome c to O2. This electron-transfer process produces a proton gradient across the membrane, which in turn drives the production of ATP. This enzyme is unique in providing energy for the cell bycoupling the electron transport through the cytochrome chain with the process of oxidative phosphorylation.
Purification Methods
The oxidase is purified by selective solubilisation with Triton X-100 and subsequently with lauryl maltoside, finally by sucrose gradient centrifugation [Li et al. Biochem J 242 417 1978]. It has also been purified by extraction in 0.02 M phosphate buffer (pH 7.4) containing 2% of cholic acid (an inhibitor which stabilises as well as solubilises the enzyme) and fractionated with (NH4)2SO4 collecting the 26-33% saturation cut and refractionating again and collecting the 26-33% saturation fraction. The pellet collected at 10,000xg appears as an oily paste. The cholate needs to be removed to activate the enzyme as follows: The precipitate is dissolved in 10mL of 0.1M phosphate buffer pH 7.4, containing 1% of Tween-80 and dialysed against 1L of 0.01 M PO4 buffer (pH 7.4) containing 1% of Tween-80 for 10hours at 0o and aliquoted. The enzyme is stable at 0o for 2 weeks and at -15o for several months. It is assayed for purity (see reference) by oxidation of reduced cytochrome c (Km 10WM). [Yonetani Biochemical Preparations 11 14 1966, J Biol Chem 236 1680 1961.] Cytokines See chemokines, interferons, interleukins. Deoxyribonucleic acid (from plasmids). These are purified by two buoyant density ultracentrifugations using ethidium bromide-CsCl. The ethidium bromide is extracted with Et2O, and the DNA is dialysed against buffered EDTA and lyophilised. [Marmur & Doty J Mol Biol 5 109 1962, Guerry et al. J Bacteriol 116 1064 1973.] See “Introduction” in Chapter 6.
