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Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.

Abstract：

:The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Robinson KM,Janes MS,Pehar M,Monette JS,Ross MF,Hagen TM,Murphy MP,Beckman JS

doi

10.1073/pnas.0601945103

subject

Has Abstract

pub_date

2006-10-10 00:00:00

pages

15038-43

issue

eissn

0027-8424

issn

1091-6490

pii

0601945103

journal_volume

103

pub_type

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Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.