K. Staniek et H. Nohl, H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system, BBA-BIOENER, 1413(2), 1999, pp. 70-80
Evaluation of the existence of superoxide radicals (O-2(.-)), the site of g
eneration and conditions required for one-e(-) transfer to oxygen from biol
ogical redox systems is a prerequisite for the understanding of the deregul
ation of O-2 homeostasis leading to oxidative stress. Mitochondria are incr
easingly considered the major O-2(.-) source in a great variety of diseases
and the aging process. Contradictory reports on mitochondrial O-2(.-) rele
ase prompted us to critically investigate frequently used O-2(.-) detection
methods for their suitability. Due to the impermeability of the external m
itochondrial 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. Tetramethylbenzi
dine-dependent H2O2 detection was counteracted by rereduction of the dye th
rough 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 b
y mitochondrial protein constituents. The present study has resolved this p
roblem by separating the detection system from H2O2-producing mitochondria.
(C) 1999 Elsevier Science B.V. All rights reserved.