H2O2 produced by mitochondria is measured by reacting it with homovanillic acid in the presence of horseradish peroxidase (see above). This reaction produces a fluorescent dimer at 312 nm excitation and 420 nm emission.
The incubation medium contains 145 mM KCl, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, and 0.1% fatty-acid free albumin at pH 7.4. The pH of the medium must be adjusted at the same temperature used during the assay (37°C). Solutions of substrates, ADP and enzymes are prepared in this medium without albumin at the following (initial) concentrations: 70 Units/ml of high purity horseradish peroxidase, 4 mM homovanillic acid, pyruvate/malate (125 mM or 250 mM depending on the tissue) and succinate neutralized to pH 7.4 (250 mM in heart and liver; 500 mM in brain).
The different components of the assay are added to tubes in the following order after adding a large volume of incubation medium: (1) mitochondria, (2) horseradish peroxidase, (3) homovanillic acid, (4) superoxide dismu-tase, and (5) the substrates (pyruvate/malate, glutamate/ malate or succinate+rotenone) to start the reaction. The volume of incubation medium added should be around 85% of the total reaction volume (e.g., 1.5 ml of total reaction volume). The volumes added of the rest of the reactants are those needed to reach the following final concentrations: 0.25 mg of mitochondrial protein per ml in heart and liver, 0.4 mg of mitochondrial protein per ml in brain, 6 U/ml of horseradish peroxidase, 0.1 mM
homovanillic acid, 50 U/ml of SOD, 5 mM succinate + 2 ^M rotenone, 2.5 mM pyruvate/2.5 mM malate or 2.5 mM glutamate/2.5 mM malate in heart and liver. However, in brain, it is advisable to increase the concentration of substrate to 10 mM for succinate (+ 2 ^M rotenone) and to 5 mM for pyruvate. At those concentrations H2O2 production is not substrate-dependent. To localize the place where ROS are produced inside the electron transport, chain inhibitors must be employed. Rotenone (2 ^M final concentration) is used to inhibit at complex I, thenoyltrifluoroacetone (TTFA, 11 ^M final concentration) for complex II, and antimycin A (10 ^M final concentration) for complex III. Solutions of inhibitors are prepared in pure high-grade ethanol. The reaction is performed during 15 minutes with constant agitation in a temperature-controlled water bath at 37°C. After 15 minutes of incubation, the reaction is stopped, the samples are transferred to an ice-cold bath, 0.5 ml of the stop buffer (2 M glycine, 2.2 M NaOH, 50 mM EDTA) is added (per each 1.5 ml of reaction volume), and the fluorescence (312 nm excitation, 420 nm emission) is measured. With the addition of the glycine-NaOH-EDTA the final pH is around 12.4, which increases the sensitivity and makes the final fluorescence essentially pH-indepen-dent. This procedure highly decreases the variability of results compared to measuring the fluorescence at neutral pH at which it is strongly pH dependent.
In the absence of SOD, the rates represent H2O2 production. SOD added in excess converts O2 produced (if any) to H2O2. Thus, in the presence of SOD, the assay estimates the mitochondrial production of O2 plus H2O2. Since the respiratory chain univalently reduces oxygen to O2 which then dismutates to H2O2, the measurement of mitochondrial H2O2 production represents the rate of oxygen radical generation. Moreover, the addition of SOD can prevent the underestimation of the rate of H2O2 production due to reaction of O2 with HRP or HRP-Compound I (Muller et al., 2004).
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