Spectroscopic Determination of Cytochrome c Oxidase Content in Tissues Containing Myoglobin or Hemoglobin

National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Analytical Biochemistry (Impact Factor: 2.22). 07/1996; 237(2):274-8. DOI: 10.1006/abio.1996.0239
Source: PubMed


A simple spectroscopic method for determining the cytochrome c oxidase, cytochrome a, a3, content in tissue and mitochondria samples independent of myoglobin or blood contamination is described. Using tissue homogenates solubilized in Triton X-100, this assay relies on the selective reduction of mitochondrial cytochromes by the action of potassium cyanide. Monitoring the optical absorbance of these samples at 605 nm provided a quantitative determination of cytochrome c oxidase content in the presence of myoglobin or blood. The cytochrome c oxidase content of porcine heart mitochondria and whole tissue was determined to be 0.85 nmol/mg protein and 30.5 nmol/g wet wt, respectively.

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    • "The samples were then reduced using excess sodium hydrosulfite, and absorbance was again measured between 510 and 630 nm. Difference spectra between the oxidized and reduced mitochondrial preparations were calculated, and cytochrome a content was determined from the peak at 605 nm using an extinction coefficient of 12 l/(mol·cm) (Balaban et al., 1996). "
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    • "Citrate synthase activity and cytochrome c oxidase levels Citrate synthase (a marker for mitochondrial content) activity was determined spectrophotometrically following the protocol of Srere (Srere, 1969). Cytochrome c oxidase (Complex IV) content was also determined spectrophotometrically, as previously described (Balaban et al., 1996). All high resolution respiration measurements were expressed as O 2 flux (picomoles O 2 per s À1 per mg wet weight) and corrected for citrate synthase (CS) activity (Rabol et al., 2009, 2010). "
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