Mammalian cytochrome-c oxidase: Characterization of enzyme and immunological detection of subunits in tissue extracts and whole cells

To explore the biogenesis of mammalian cytochrome-c oxidase and to examine the defects of this enzyme in human disease, approaches are required to measure the hemes spectrally and assay enzyme activity in tissue extracts. This chapter discusses these approaches. It is important to be able to characterize the subunit composition of the enzyme to look for alterations in any subunit based on their migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels, absence of any subunit, and incomplete assembly. The panel of monoclonal antibodies for such studies and their use is described. Several of the monoclonal antibodies will be useful in isolation of the cytochrome-c oxidase complex by immuno-affinity chromatography after covalent binding to Sepharose CL-4B (Pharmacia LKB Biotechnology). This method is established for the purification of the yeast enzyme, using monoclonal antibodies to yeast subunit III, and a similar method is developed for the purification of the human enzyme. © 1995 Elsevier Inc.