Time-resolved optical spectroscopy on intact myocytes

Abstract

Myocytes prepared from perfused rat heart were studied spectroscopically using a photodiode array spectrophotometer adapted to a rapid mixing stopped-flow apparatus. The isolated cells were found to be viable for 3 to 4 hours, i.e. over the total time of the experiments. Sodium ascorbate and tetramethyl-para-phenylenediamine were used as exogenous reductants. Cytoplasmic and mitochondrial membranes were found to be freely permeable to tetramethyl-para-phenylenediamine. The use of singular value decomposition proved to be powerful in resolving the spectral contributions of the chromophoric components within the overall absorption spectrum. Spectral resolution was improved by adding carbon monoxide at a concentration that kept myoglobin fully saturated without affecting the activity of cytochrome c oxidase. The redox state of cytochrome c and cytochrome a was observed during the steady-state consumption of oxygen and during the reduction following the exhaustion of oxygen. The redox state of the two chromophores was found to be approximately equal and close to 25-30% oxidized during steady-state respiration; during the final reduction they changed simultaneously. These experiments suggest that in living cells, as in the purified enzyme, the rate limiting step of the turnover of cytochrome oxidase is the internal transfer of electrons from cytochrome a to cytochrome a3.