Triton X-100 Induced Dissociation of Beef Heart Cytochrome c Oxidase into Monomers

Neal C. Robinson, Linda Talbert

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Abstract

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200000, a Stokes radius of 66–72 Å, and an S020,w= 8.70 S. These values were determined by (1) measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, (2) evaluation of the sedimentation coefficient, S020,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and (3) measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since (1) the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and (2) a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer. These results suggest that homogeneous, dimeric, Triton X-100 solubilized cytochrome c oxidase is very difficult to isolate as a stable species.

Original languageEnglish (US)
Pages (from-to)2328-2335
Number of pages8
JournalBiochemistry
Volume25
Issue number9
DOIs
StatePublished - May 1986

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ASJC Scopus subject areas

  • Biochemistry

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