Measurement of mitochondrial oxygen consumption using a Clark electrode

Abstract

Mitochondria require oxygen to produce ATP in sufficient quantities to drive energy-requiring reactions in eukaryotic organisms. The measurement of oxygen consumption rates from isolated mitochondria in vitro is a useful and valuable technique in the research and evaluation of mitochondrial dysfunction and disease since ADP-dependent oxygen consumption directly reflects coupled respiration or oxidative phosphorylation (OXPHOS). This chapter describes the traditional method of mitochondrial polarography using a Clark electrode for measuring coupled respiration in freshly isolated mitochondria from both mammalian tissues and Drosophila melanogaster.

Fig. 1.

Idealized trace of a polarographic run. At time “0,” substrate (5 μM of glutamate + malate or succinate) is added to chamber full of air-saturated respiration buffer, and a stable baseline is observed. (a) Isolated mitochondria are added to a final concentration of 0.3 mg/mL. (b) 125 nmol of ADP is added, stimulating state III rate which transitions to state IV once exogenous ADP is consumed by OXPHOS. (c) DNP (50 μM) is added to stimulate maximal uncoupled rate (UC).

Fig. 2.

Typical polarographic trace of rat heart mitochondria with succinate as substrate. A polarographic experiment using mitochondria isolated from wild-type rat heart is shown. (a) Succinate (5 μM) is added. (b) Rat heart mitochondria (0.9 mg/mL) is added. (c) 125 nmol of ADP is added. (d) DNP (50 μM) is added.

Fig. 3.

Typical polarographic trace of Drosophila larval mitochondria with glutamate + malate as substrate. A polarographic experiment using mitochondria isolated from wild-type Drosophila melanogaster third instar larvae is shown. (a) Glutamate + malate (5 μM each) is added. (b) Fly larval mitochondria (0.3 mg/mL) is added. (c) 125 nmol of ADP is added. (d) DNP (50 μM) is added.