Superoxide released into the mitochondrial matrix.
Reactive oxygen species (ROS) that are produced by mitochondria are released toward the mitochondrial matrix or the intermembrane space. Each ROS pool is likely involved in different cellular mechanisms and damage. Unfortunately, it is difficult to distinguish the provenance and effects of ROS. Here we introduce a method to semiquantitate the steady-state levels of superoxide produced in the matrix of mitochondria. Superoxide produced during cellular respiration is capable of oxidizing hydroethidine, a probe that is membrane permeant. The poor membrane permeability of the hydroethidine oxidation products causes accumulation of these fluorescent products within the mitochondria. After isolation of mitochondria, a method based on the capillary electrophoretic separation of individual organelles and their detection by laser-induced fluorescence detection is used to determine their fluorescent contents. Use of this method for the analysis of organelle fractions obtained from cells treated with antimycin A or rotenone confirms that the detected fluorescence is associated with superoxide produced by mitochondria. Furthermore, using this method the superoxide levels in the mitochondrial matrix of a cytoplasmic hybrid (cybrid) cell line (DeltaH2-1) and one of its parent cell lines (143B) were compared.
Meany
Danni L
DL
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Poe
Bobby G
BG
Navratil
Marian
M
Moraes
Carlos T
CT
Arriaga
Edgar A
EA
D000447:Aldehydes / Q000494:pharmacology; D001859:Bone Neoplasms; D002458:Cell Fractionation / Q000379:methods; D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D012516:Osteosarcoma; D010617:Phenanthridines / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*
D000447:Aldehydes; D004272:DNA, Mitochondrial; D010617:Phenanthridines; C111472:mitotracker green FM; D013481:Superoxides; C058428:hydroethidine