Measurement of ROS homeostasis in isolated mitochondria

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Citations (Scopus)

Abstract

In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages199-223
Number of pages25
EditionC
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Enzymology
NumberC
Volume547
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Aconitase
  • Amplex
  • Mitochondria
  • Oxidative stress
  • Reactive oxygen species
  • Red
  • ROS elimination

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Tretter, L., & Ambrus, A. (2014). Measurement of ROS homeostasis in isolated mitochondria. In Methods in Enzymology (C ed., pp. 199-223). (Methods in Enzymology; Vol. 547, No. C). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-801415-8.00012-6