Fig. 1. A typical oxidative phosphorylation showing oxygen consumption (down y-axis) over time (x-axis). To start, the chamber is filled with 500 μl halothane-treated buffer. Then, 500 μg mitochondria and a small amount of adenosine diphosphate (ADP) (to allow metabolizing of internal substrates if they are present—here, not the case) are added. Next, the complex I–dependent substrate malate is added. (In other experiments, succinate is added as a substrate for complex II.) Finally, 85 nmol ADP is added to allow oxidative phosphorylation to occur. State 3 is the fast respiration in response to ADP. When the ADP is all phosphorylated and becomes limiting, respiration slows to state 4. As an example for all rate determinations, the top inset depicts the calculation of a state 3 respiration rate. The lower inset shows the ADP/O ratio, calculated as the consumption of ADP added divided by the amount of oxygen consumed during state 3 respiration. These measurements are repeated a second time. A much higher amount of ADP is then added to ensure that ADP was not limiting in the state 3 measurements. The uncoupler 2,4-dinitrophenol (DNP) is added to determine whether phosphorylation of ADP was limiting for respiration. At the end of the experiment, N,N,N,N-tetramethyl-p-phenylenediamine (TMPD)–ascorbate is added as a direct electron donor to cytochrome c. The ensuing respiration rate depends only on the amount of intact mitochondria present and is therefore used as an internal loading control.