Differential detergent solubility investigation of thermally induced transitions in cytochrome c oxidase.
The thermal denaturation of membrane-reconstituted cytochrome c oxidase (EC 1.9.3.1) occurs at approximately 63 degrees C as determined by high-sensitivity differential scanning calorimetry. The heat capacity profile associated with this process is characterized by the presence of two well-defined peaks, indicating that all the enzyme subunits do not have the same thermal stability. This thermal denaturation of the enzyme complex is coupled to a change in its solubility properties. This change in solubility allows separation of the native and denatured protein fractions by detergent solubilization followed by centrifugation under conditions in which only the native fraction is solubilized. Using this principle, it has been possible to study the denaturation of membrane-reconstituted cytochrome c oxidase and quantitatively identify the protein subunits undergoing thermal denaturation using computer-assisted gel electrophoresis analysis. This technique allows calculation of single-subunit thermal denaturation profiles within the intact enzyme complex, and as such, it can be used to obtain transition temperatures, molecular populations, and van't Hoff enthalpy changes for individual protein subunits, thus complementing results obtained by high-sensitivity differential scanning calorimetry.
Rigell
C W
CW
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Freire
E
E
D000818:Animals; D002417:Cattle; D003720:Densitometry; D003902:Detergents; D004134:Dimyristoylphosphatidylcholine; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007834:Lasers; D008081:Liposomes; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D011489:Protein Denaturation; D012995:Solubility; D013501:Surface-Active Agents; D013816:Thermodynamics
D003902:Detergents; D008081:Liposomes; D046911:Macromolecular Substances; D013501:Surface-Active Agents; D003576:Electron Transport Complex IV; D004134:Dimyristoylphosphatidylcholine