The utilisation of oxygen during the reaction is measured using an oxygen electrode, which determines the percentage saturation of oxygen in the reaction mixture. The instrument is calibrated against a buffer saturated with oxygen to set 100% saturation and a buffer totally depleted of oxygen (by reaction with a strong reducing agent such as sodium dithionite) to set 0% saturation.
The reaction chamber is separated from the electrodes by a teflon membrane, which permits oxygen to diffuse from the reaction buffer into the potassium chloride solution that bathes the electrodes: a platinum cathode and a silver anode. A voltage is applied between the electrodes and the resulting current (approx. 1 µA) is proportional to the concentration of oxygen.
At the cathode oxygen is reduced to water:
At the anode metallic silver is oxidised to silver chloride:
After allowing the buffer and substrate to equilibrate, the mitochondrial preparation is added through the injection port, and the consumption of oxygen is measured for a short time, then the ADP and any inhibitors, etc, are added, and oxygen consumption measured until there is no further reaction. The results from a typical experiment are shown here:
In the electrode you will be using in these studies,
100% saturation with oxygen = 1327 nmol O.