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Fluorogenic Analysis of H2O2 in Biological Materials

  • Kenneth Hensley
  • Kelly S. Williamson
  • Robert A. Floyd
Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

The biological importance of hydrogen peroxide and other “reactive oxygen species” (ROS) has become greatly appreciated in recent years. It has become apparent that certain ROS, in particular hydrogen peroxide (H2O2) and nitric oxide (•NO), are ubiquitously used as intra- or intercellular messengers (1). Because these ROS are short-lived in vivo, their steady-state concentrations remain low, and their accurate quantitation poses a significant technical challenge. Moreover, it is often necessary to monitor changes in ROS in real time, for instance during the time course of hormonal stimulation of cultured cells (2). The most common methods of measuring H2O2 rely upon peroxide-dependent oxidation of reduced xanthene dyes such as 2′,7′-dichlorodihydrofluorescein (H2DCF, also called dichlorofluorescin) or dihydrorhodamine 123 (H2RD123) (Fig. 1). These dyes were originally used to measure peroxidase activities (3) but the assays were easily modified to allow peroxide determination (2,4, 5, 6, 7). Although the reduced dyes are not highly fluorescent, their oxidation products are, and can be monitored continuously using a fluorescence spectrometer, microplate reader, or confocal microscope. Although flexible and convenient, the fluorogenic determination of H2O2 must be performed with due consideration of factors that may interfere with the chemistry of the assay.
Fig. 1.

Chemistry of H2DCFDA oxidation using H2O2 as a terminal electron acceptor.

Keywords

Reactive Oxygen Species Esterase Activity H2O2 Generation Acetoxymethyl Ester Accurate Quantitation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Sen, C. K. and Packer, L. (1996) Antioxidant and redox regulation of gene transcription. FASEB J. 10, 709–720.PubMedGoogle Scholar
  2. 2.
    Robinson, K. A., Stewart, C. A., Pye, Q. N., Nguyen, X., Kenney, L., Salzman, S., et al. (1999) Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture. J. Neurosci. Res. 55, 724–732.PubMedCrossRefGoogle Scholar
  3. 3.
    Brandt, R. and Keston, A. S. (1965) Synthesis of diacetyldichlorofluorescin: A stable reagent for fluorometric analysis. Anal. Biochem. 11, 6–9.PubMedCrossRefGoogle Scholar
  4. 4.
    Loschen, G., Azzi, A., and Flohe, L. (1973) Mitochondrial H2O2 formation: Relationship with energy conservation. FEBS Lett. 33, 84–88.PubMedCrossRefGoogle Scholar
  5. 5.
    Baggiolini, M., Ruch, W., and Cooper, P. H. (1986) Measurement of hydrogen peroxide production by phagocytes using homovanillic acid and horseradish peroxidase. Methods Enzymol. 132, 395–400.PubMedCrossRefGoogle Scholar
  6. 6.
    LeBel, C. P., Ischiropoulos, H., and Bondy, S. C. (1992) Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231.PubMedCrossRefGoogle Scholar
  7. 7.
    Royall, J. A. and Ischiropoulos, H. (1993) Evaluation of 2′,7′-dichloro-fluorescin and dihydrorhodamine as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302, 348–355.PubMedCrossRefGoogle Scholar
  8. 8.
    Ajtai, K. and Burghardt, T. P. (1992) Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into π-radicals by photochemical reduction. Biochemistry 31, 4275–4282.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc.,Totowa, NJ 2003

Authors and Affiliations

  • Kenneth Hensley
    • 1
  • Kelly S. Williamson
    • 1
  • Robert A. Floyd
    • 1
  1. 1.Free Radical Biology and Aging ProgramOklahoma Medical Research FoundationOklahoma City

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