title: Mitochondrial thioredoxin-responding off-on fluorescent probe.
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) × 10(3) (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.
authors.affiliation: Department of Chemistry, Korea University, Seoul, 136-701, Korea.
authors.forename: Min Hee
authors.initials: MH
authors.lastname: Lee
authors.forename: Ji Hye
authors.initials: JH
authors.lastname: Han
authors.forename: Jae-Hong
authors.initials: JH
authors.lastname: Lee
authors.forename: Hyo Gil
authors.initials: HG
authors.lastname: Choi
authors.forename: Chulhun
authors.initials: C
authors.lastname: Kang
authors.forename: Jong Seung
authors.initials: JS
authors.lastname: Kim
mesh_terms: D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry*; D015394:Molecular Structure; D013879:Thioredoxins / Q000737:chemistry*; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D013879:Thioredoxins