An indirect and highly sensitive method for the determination of the flow of reducing equivalents in the respiratory chain.

Reactions leading to oxido-reduction of TMPD have shown that, in its oxidized form, this compound has among others an extinction maximum at 610 nm; with the exception of cytochrome a, at this wavelength none of the respiratory chain intermediates has the ability to absorb the incident light. This property together with the one of reacting with cytochrome c, has given us the possibility to use TMPD as a "probe" of the reducing equivalents flow in the respiratory chain. Added to mitochondrial suspension, TMPD undergoes redox cycles in relation to the activity of the respiratory chain, modulated by increasing concentrations of succinate or respiratory inhibitors. NEM-induced reversible oxidation on the respiratory intermediates can also be determined by following the TMPD oxidation. The preliminary data obtained are thus consistent with the hypothesis that in appropriate conditions, the TMPD redox state can be used as a probe of the respiratory chain activity.