Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases

Background: The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for measuring oxidants released extra-cellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. Scope of review: Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O-2(radical anion) and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. Major conclusions: Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. Several reliable methods for measurement of extracellular O-2(radical anion) and H2O2 by NOX proteins are available. Chemiluminescent probes for both extracellular and intracellular O-2(radical anion) and H2O2 detection have shortcomings that limit their use. Options for quantitation of intracellular O-2(radical anion) and H2O2 are very limited. However, non-redox sensitive probes and genetically encoded reporters promise to provide spatial and temporal detection of O-2(radical anion) and H2O2. General significance: The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. This article is part of a Special Issue entitled Current methods to study reactive oxygen species-pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. (C) 2013 Elsevier B.V. All rights reserved.