A microspectrophotometric quantification of cytochrome oxidase activity by means of color-modified diaminobenzidine reaction.

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  • HIRAOKA TOSHISUKE
    School of Medicine, Shiga University of Medical Science Biological Laboratory, Osaka University of Commerce
  • HIRAI KEI-ICHI
    Department of Anatomy, Kanazawa Medical University
  • UYEDA TADASHI
    Department of Anatomy, Kanazawa Medical University

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A new microspectrophotometric method for quantifying cytochrome oxidase activity has been developed based on a color-modified diaminobenzidine (DAB) reaction. The reaction consists of pretreating tissue sections or cell smears with 10mM cobalt acetate dissolved in 0.1M tris-HCl buffer of pH 7.4 for 10min at 37°C, washing and then treating with the DAB reagent for cytochrome oxidase for 15min at 37°C. Mitochondria turn reddish blue in the stained sections or smears. This reaction is enzymatic and conforms to Michaelis-Menten's formula. The microspectrophotometric quantification is effected by scanning the selected cytoplasmic area at 575nm or by measuring the cytoplasmic plug at two wavelengths, 575 and 665nm. As an example, the monomodal distribution in cytochrome oxidase activity was demonstrated among isolated rat liver cells by the two wavelength method.

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