A valid quantitative histochemical technique for assaying cytochrome c oxidase in single cells

A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ.