Mitochondrial thioredoxin-responding off-on fluorescent probe

Min Hee Lee, Ji Hye Han, Jae Hong Lee, Hyo Gil Choi, Chulhun Kang, Jong Seung Kim

Research output: Contribution to journalArticle

118 Citations (Scopus)

Abstract

We synthesized a new probe, Mito-Naph, to visualize mitochondrial thioredoxin (Trx) activity in cells. A fluorescence off-on change is induced by disulfide cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular cyclization by the released thiolate to give a fluorescent product. By measuring the fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to 50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for Trx is (4.04 ± 0.26) × 103 (M s) -1, approximately 5000 times faster than that for GSH. The inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with other biological thiols containing amino acids in vitro and in vivo.

Original languageEnglish
Pages (from-to)17314-17319
Number of pages6
JournalJournal of the American Chemical Society
Volume134
Issue number41
DOIs
Publication statusPublished - 2012 Oct 17

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

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