Reaction of superoxide with trityl radical: Implications for the determination of superoxide by spectrophotometry

Vijay Kumar Kutala, Narasimham L. Parinandi, Jay L. Zweier, Periannan Kuppusamy

Research output: Contribution to journalArticle

Abstract

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1×103M-1s-1. In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.

Original languageEnglish (US)
Pages (from-to)81-88
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume424
Issue number1
DOIs
StatePublished - Apr 1 2004
Externally publishedYes

Fingerprint

Spectrophotometry
Superoxides
Assays
Electron Spin Resonance Spectroscopy
Cytochromes c
Oxidation-Reduction
Paramagnetic resonance
Hydrogen Peroxide
Spectrum Analysis
Spectroscopy
Redox reactions
Reducing Agents
Biological Factors
Oxidants
Free Radicals
Glutathione
Rate constants

Keywords

  • Cytochrome c
  • Spectrophotometry
  • Superoxide
  • Trityl radical
  • Xanthine oxidase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Reaction of superoxide with trityl radical : Implications for the determination of superoxide by spectrophotometry. / Kutala, Vijay Kumar; Parinandi, Narasimham L.; Zweier, Jay L.; Kuppusamy, Periannan.

In: Archives of Biochemistry and Biophysics, Vol. 424, No. 1, 01.04.2004, p. 81-88.

Research output: Contribution to journalArticle

Kutala, Vijay Kumar ; Parinandi, Narasimham L. ; Zweier, Jay L. ; Kuppusamy, Periannan. / Reaction of superoxide with trityl radical : Implications for the determination of superoxide by spectrophotometry. In: Archives of Biochemistry and Biophysics. 2004 ; Vol. 424, No. 1. pp. 81-88.
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