Spectral and electron transfer properties of Sepharose 6MB-immobilized cytochrome c.
Cytochrome c has been covalently attached to Sepharose 6MB to study the effects of immobilization on the molecule. A detailed study of the resulting product was conducted based on its three characteristic properties: spectra, oxidation-reduction potential, and biological activity. The spectral properties demonstrated that cytochrome c was essentially the same after attachment. No major conformational changes were indicated. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were generally 20-25 mV lower than native cytochrome c (270 mV). Heavily loaded samples, however, showed no difference in potential. The Km values for immobilized cytochrome c with cytochrome oxidase and reductase from submitochondrial particles were comparable to the soluble protein. Vmax values are more strongly affected by immobilization, especially for the reductase. It has been demonstrated that the submitochondrial particles cannot penetrate the pores of the support material and therefore only the cytochrome c molecules on the surface are available for reaction. As a support material, Sepharose 6MB, which is CNBr activated, hydrolyzes at a significant rate at the pH of the coupling reaction, and this must be considered in establishing coupling conditions for protein immobilization.
D000818:Animals; D003579:Cytochrome Reductases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D009206:Myocardium; D010084:Oxidation-Reduction; D012685:Sepharose / Q000031:analogs & derivatives; D013053:Spectrophotometry
D003574:Cytochrome c Group; D004800:Enzymes, Immobilized; C039745:Sepharose 6MB; D012685:Sepharose; D003579:Cytochrome Reductases; D003576:Electron Transport Complex IV