Effect of AF or DNCB on H₂O₂ emission from heart mitochondria under FET at states 4 and 3 of respiration. (A–C) Freshly isolated mitochondria (∼100 µg mitochondrial protein [prot.]) from mouse, rat, and guinea pig hearts were preincubated in the absence or the presence of the indicated concentrations of AF (left graphs) or DNCB (right graphs) in the presence of the NADH-linked substrates G/M (5 mM each). Monitoring of H₂O₂ was performed with the Amplex red assay during states 4 (with G/M) and 3 (+1 mM ADP) of mitochondrial respiration (Stanley et al., 2011). The specific fluxes of H₂O₂ emission are shown. The kinetic parameters describing the fluxes of H₂O₂ emission as a function of the inhibitor concentration, V_max and K_0.5, were determined after nonlinear regression fitting of the experimental points with a hyperbolic Michaelis–Menten or Hill type of equation. The results ± SEM obtained from two experiments with duplicates in each are represented.