H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system

Evaluation of the existence of superoxide radicals (O-2(.-)), the site of generation and conditions required for one-e(-) transfer to oxygen from biological redox systems is a prerequisite for the understanding of the deregulation of O-2 homeostasis leading to oxidative stress. Mitochondria are increasingly considered the major O-2(.-) source in a great variety of diseases and the aging process. Contradictory reports on mitochondrial O-2(.-) release prompted us to critically investigate frequently used O-2(.-) detection methods for their suitability. Due to the impermeability of the external mitochondrial membrane for most constituents of O-2(.-) detection systems we decided to follow the stable dismutation product H2O2. This metabolite was earlier shown to readily permeate into the cytosol. With the exception of tetramethylbenzidine none of the chemical reactants indicating the presence of H2O2 by horseradish peroxidase-catalyzed absorbance change were suited due to solubility problems or low extinction coefficients. Tetramethylbenzidine-dependent H2O2 detection was counteracted by rereduction of the dye through e(-) carriers of the respiratory chain. Although the fluorescent dyes scopoletin and homovanillic acid were found to be suited for the detection of mitochondrial H2O2 release, fluorescence change was strongly affected by mitochondrial protein constituents. The present study has resolved this problem by separating the detection system from H2O2-producing mitochondria. (C) 1999 Elsevier Science B.V. All rights reserved.