Figure 2. Measurement of H₂O₂ production by isolated mitochondria.

The production of O₂^•− within the mitochondrial matrix, intermembrane space and outer membrane leads to the formation of H₂O₂ from SOD-catalysed dismutation. Some O₂^•− can react directly with nitric oxide (NO^•) to form peroxynitrite (ONOO⁻). There are also sources outside mitochondria that produce H₂O₂ directly. The H₂O₂ efflux from mitochondria can be measured following reaction with a non-fluorescent substrate such as Amplex Red in conjunction with horseradish peroxidase (HRP) to form a fluorescent product, resorufin. Within mitochondria H₂O₂ is degraded by glutathione peroxidases (GPx) or peroxiredoxins (Prx) which depend on glutathione (GSH) and thioredoxin-2 (Trx) for their reduction respectively. Glutathione disulfide (GSSG) is reduced back to GSH by glutathione reductase (GR). Trx is reduced by thioredoxin reductase-2 (TrxR). Both enzymes receive reducing equivalents from the NADPH pool, which is kept reduced by the Δp-dependent transhydrogenase (TH), and by isocitrate dehydrogenase (ICDH). Note that many mitochondrial preparations, particularly those from the liver, contain large amounts of catalase contamination. The effects of such extramitochondrial H₂O₂ sinks are not indicated here as they are usually accounted for by appropriate H₂O₂ calibration curves in the presence of mitochondria. ox, oxidized; red, reduced. An animated version of this Figure can be seen at http://www.BiochemJ.org/bj/417/0001/bj4170001add.htm.