title: Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene.
abstract: Cytochrome c oxidase was treated with 1,5-difluoro-2,4-dinitrobenzene at molar ratios (DFDNB:oxidase) varying from 5 to 625. At the lowest ratio, there was virtually no effect of the probe on oxidase activity or on migration of oxidase subunits on sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis. At ratios of 25 and greater, there was loss of oxidase activity and a change of the pattern of subunit migration on sodium dodecyl sulfate electrophoresis. (i) Activity loss was probably a result of severely perturbing the cytochrome c binding site since oxidase activity with a low molecular weight reductant (N,N,N',N'-tetramethylphenylenediamine) was unaltered. Also unaltered were the oxidized, reduced, and carbon monoxide binding spectra of the treated oxidase. (ii) The staining pattern on sodium dodecyl sulfate electrophoresis showed that subunits III and VI disappeared from their normal positions on the gel. A new band of higher molecular weight accompanied their loss from the gel indicating that the two subunits were being cross-linked. Subunits III and VI are thus shown to have two reactive groups within 4.8 A (1 A = 0.1 nm) of one another. This proximity has not been detected with other probes that react with the same groups.
mesh_terms: D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D003576:Electron Transport Complex IV; D046911:Macromolecular Substances; D009578:Nitrobenzenes / Q000031:analogs & derivatives
chemical_list: D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009578:Nitrobenzenes; D004139:Dinitrofluorobenzene; D003576:Electron Transport Complex IV; C002041:1,5-difluoro-2,4-dinitrobenzene