Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa.
Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10-13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits. A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G-100, Bio Gel P-60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required. The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.
D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000302:isolation & purification; D046911:Macromolecular Substances; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*
D046911:Macromolecular Substances; D006418:Heme; D003576:Electron Transport Complex IV