Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Abstract

Hydrogen peroxide (H 2O 2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H 2O 2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H 2O 2 in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3'5'-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H 2O 2, methods based on dihydro compounds such as 2',7'-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H 2O 2. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H 2O 2-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H 2O 2 in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H 2O 2 can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H 2O 2, they cannot be used to monitor transient changes in H 2O 2 concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H 2O 2 and can be targeted to various compartments of the cell, but they are not selective for H 2O 2 because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H 2O 2-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR. © 2010 The Korean Society for Molecular and Cellular Biology and Springer Netherlands.