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

Title
Methods for detection and measurement of hydrogen peroxide inside and outside of cells
Authors
Rhee S.G.Chang T.-S.Jeong W.Kang D.
Ewha Authors
이서구정우진창동신강동민
SCOPUS Author ID
이서구scopusscopus; 정우진scopus; 창동신scopus; 강동민scopus
Issue Date
2010
Journal Title
Molecules and Cells
ISSN
1016-8478JCR Link
Citation
vol. 29, no. 6, pp. 539 - 549
Indexed
SCI; SCIE; SCOPUS; KCI WOS scopus
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.
DOI
10.1007/s10059-010-0082-3
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일반대학원 > 생명·약학부 > Journal papers
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