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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Available from: Robert S Balaban
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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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    • "Another challenge is the determination of low amounts against a highly concentrated matrix (e.g. high purity reagents [3,4] or trace components in biological samples against a highly absorbing matrix like hemoglobin or albumin [5,6]), which should also be characterized in detail, and with minimum pretreatment. As the signal of the main component overwhelms the other, the applicability of electroanalysis or chromatography is sometimes hindered [7,8], and thus require sophisticated separation/isolation techniques. "
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    • "Mitochondrial content in Mb KO and WT tissues was evaluated as the amount of cytochrome a,a 3 (cyt a) present. Cyt a content was determined in tissue homogenates as described previously [7] for three paired Mb KO and WT hearts and four paired Mb KO and WT TA muscles. "
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