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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
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Oxidative modifications of mitochondria complex II

Liwen Zhang1, Patrick T Kang, Chwen-Lih Chen

  • 1Proteomics and Mass Spectrometry Facility, Campus Chemical Instrument Center, The Ohio State University, Columbus, OH, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 23, 2013
PubMed

Insights

Mitochondrial dysfunction in heart attacks involves oxidative damage to Complex II. This study details methods to identify specific oxidative modifications, aiding understanding of heart injury mechanisms.

Area of Science:

  • Biochemistry
  • Cardiovascular Biology
  • Mitochondrial Biology

Background:

  • Mitochondrial dysfunction, driven by superoxide (O2·−) and nitric oxide (NO), is central to myocardial ischemia/reperfusion injury.
  • Oxidative impairment in Complex II involves decreased S-glutathionylation and increased tyrosine nitration of its 70 kDa subunit in post-ischemic hearts.

Purpose of the Study:

  • To investigate Reactive Oxygen Species (ROS)-mediated oxidative modifications in Complex II relevant to myocardial infarction.
  • To describe a protocol for characterizing specific oxidative modifications at the Complex II 70 kDa subunit.

Main Methods:

  • In vitro oxidative modification of isolated Complex II using GSSG and OONO−.
  • Nano-liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) for detailed analysis of modified subunits.
  • Immunofluorescence microscopy using specific antibodies to detect protein nitration and S-glutathionylation in myocytes under oxidative stress.

Main Results:

  • Established a protocol for identifying specific oxidative modifications on the Complex II 70 kDa subunit.
  • Demonstrated the feasibility of detecting cellular oxidative modifications, including protein nitration and S-glutathionylation, using immunofluorescence microscopy.

Conclusions:

  • The described methods enable precise characterization of oxidative damage to Complex II.
  • These techniques are valuable for studying ROS-mediated mechanisms in myocardial infarction and related conditions.

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