Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay

Abstract

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125-750 μM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria.

Keywords: Amplex Red; Hydrogen peroxide; Mitochondria; Resorufin; Respiratory chain; Respiratory complex I.

Graphical abstract

Fig. 1

An example of determination of oxygen concentration in the standard reaction mixtures used throughout this study. Uncoupled (2 μM FCCP was added) oxidation of 5 mM succinate was traced as fumarate formation determined at 278 nm $(\mathrm{\epsilon }\frac{\mathrm{mM}}{278}=0.3)$. The reaction was initiated by the addition of SMP (0.15 mg of protein per ml) (indicated by arrows) to the closed spectrophotometric cuvette with air saturated (curve 1) and standard reaction mixture saturated by pure oxygen (curve 2). Figures on the traces in italic are the specific oxidase activities (μmol/min per mg of protein). Practically no fumarate formation was seen in a reaction mixture saturated by pure argon.

Fig. 2

Initial rates of Res formation by coupled SMP (40 μg/ml) oxidizing (A), succinate (5 mM); (B), succinate (5 mM) and NADH (1 mM); and (C), succinate (50 μM) in the presence of 5 μM rotenone and 1.6 μM myxothiazol. Rotenone (5 μM) was added where indicated. Open triangles on line 1 in panel (A) depict the rates determined from the progress curve of Res formation as described in Fig. 5A.

Fig. 3

Initial rates of Res formation by intact rat heart mitochondria (50 μg/ml) oxidizing (A), glutamate and malate (5 mM each); (B), succinate (5 mM); (C), succinate (50 μM) in the presence of 5 μM rotenone and 1.6 μM myxothiazol. Rotenone (5 μM) and malonate (5 mM) were added where indicated.

Fig. 4

Initial rates of Res formation by permeabilized rat heart mitochondria. Mitochondria (30 μg/ml) were preincubated in the standard reaction mixture for 1 min in the presence of alamethicin (40 μg/ml) and 1 mM MgCl₂. The reaction was initiated by the addition of 1 mM NADH. Where indicated mitochondria after permeabilization were preincubated with NADH-OH (70 nM) for 30 s. Ammonium chloride (30 mM) was added to the samples where indicated.

Fig. 5

Time course of hydrogen peroxide production by coupled SMP (0.2 mg/ml) oxidizing succinate. (A), line 1, oxygen consumption initiated by 5 mM succinate (S) as measured amperometrically; line 2, Res formation; line 4, rotenone (5 μM) and NADH (100 μM) (indicated by arrow) were added after all oxygen was consumed (anaerobic conditions); line 3, NADH-OH (0.2 μM) was added before NADH; line 5 in red, calculated trace described by Eq. (4) (see text) with parameters: k₁ = 0.0014 min^–1 (first-order initial rate dependence depicted in Fig. 2A) and k₂ = 30 μM/min (from zero-order rate constant of oxygen consumption, line 1). Calibration of the scale by additions of hydrogen peroxide (P, 0.5 μM each). (B), instant rates of the reaction obtained from derivatization of actual trace of Res formation shown in panel (A) plotted as a function of oxygen concentration. (C), initial rates of Res formation as a function of assay time. Aliquots (0.4 ml) were withdrawn from the sample assayed as depicted in panel (A), added to 1.6 ml of the standard reaction mixture supplemented by AR, HP, SOD, and 5 mM succinate, and the initial rates of hydrogen peroxide formation were measured. Arbitrary unit (1.0) corresponds to the specific rate of 1.4 nmol per min per mg.

Fig. 6

Time course of Res formation during oxidation of succinate by intact rat heart mitochondria (0.29 mg/ml). (A), actual traces of oxygen consumption (curve 1) and Res formation (curves 2–4). Kinetics of Res disappearance after the sample became anaerobic (indicated by arrow) are depicted by curves: 3, no further additions; 4, alamethicin (40 μg/ml) and MgCl₂ (1 mM) were added; 2, as 4, 0.2 μM NADH-OH was added; line 5 in red, calculated trace described by Eq. (4) (see text) with the parameters: k₁ = 0.0011 min^–1 (first-order initial rate dependence depicted in Fig. 3B) and k₂ = 33 μM/min (from zero-order rate constant of oxygen consumption, line 1). (B), instant rates of the reaction obtained from derivatization of actual trace of Res formation shown in panel (A) plotted as a function of oxygen concentration. (C), The initial rates of Res formation as a function of the assay time; see details in Fig. 5C. Arbitrary unit (1.0) corresponds to the specific rate of 1.0 nmol per min/mg.

Fig. 7

Reversible redox transformation of Res. (A) Res (4 μM) was added to the standard reaction mixture (2.4 ml) supplemented by SMP (0.4 mg/ml), succinate (5 mM), and rotenone (5 μM), and the mixture was incubated in a closed cuvette until all oxygen was consumed. NADH (100 μM) was added where indicated, and decolorization of Res was traced (curve 2). The cuvette was opened, stirred where indicated, closed, and further decolorization continued (curve 2). No change in absorbance was detected in samples where no NADH was added (curve 1). (B), Res was decolorized (reduced by NADH) as in panel (A), and a small amount of oxygen (10 μM, 0.1 ml of aerobic aqueous solution containing 25 mM potassium cyanide to prevent respiratory activity of SMP) and restoration of absorbance was followed (curve 1). E. coli SOD (cyanide-insensitive, 24 units/ml) was added (curve 2). ResH₂ was recolorized by hydrogen peroxide (10 μM) (curves 3 and 4). HP (0.5 units/ml) was added where indicated (curve 3) or before hydrogen peroxide was added (curve 4).