Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.
We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.
D000818:Animals; D002417:Cattle; D002697:Chloral Hydrate; D003576:Electron Transport Complex IV; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; D014508:Urea
D012967:Sodium Dodecyl Sulfate; D002697:Chloral Hydrate; D014508:Urea; D003576:Electron Transport Complex IV; D014357:Trypsin