Superoxide released into the mitochondrial matrix

Danni L. Meany, Bobby G. Poe, Marian Navratil, Carlos T. Moraes, Edgar A. Arriaga

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


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 (ΔH2-1) and one of its parent cell lines (143B) were compared.

Original languageEnglish (US)
Pages (from-to)950-959
Number of pages10
JournalFree Radical Biology and Medicine
Issue number6
StatePublished - Sep 15 2006

Bibliographical note

Funding Information:
This work was supported by grant from the National Institutes of Health (R01-AG-20866).


  • Heteroplasmy
  • Hydroethidine
  • Matrix
  • Mitochondria
  • Superoxide


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