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.

Toward a Chemical Marker for Inflammatory Disease: A Fluorescent Probe for Membrane-Localized Thioredoxin

“…18,23 As can be seen from an inspection of the upper panels of Figure 3, the FI of the HeLa cells treated with 1 decreases in a PX-12 dose-dependent manner. In contrast, the fluorescence from 12 is unchanged under similar experimental conditions (lower panels in Figure 3).…”

“…For instance, we previously reported a fluorescent probe that can visualize mitochondrial Trx activity (as opposed to membrane-localized Trx activity) in living cells. 18 This system proved highly specific for Trx. Thus, building off this prior work, we have developed a new membrane-targeted Trx-specific fluorescent probe.…”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…The same group of researchers described a probe bearing triphenylphosphinenaphthalimide with a disulfide moiety in order to measure the activity of the mitochondrial thioredoxin (Trx) level in cells. Such information may also indicate illnesses related to cancer [123].…”

Nanophotonics and supramolecular chemistry

“…Detection systems for small organic molecules accompanied by selective chemical reactions in cells have also been developed. For example, Kang, Kim, and coworkers reported an off-on fluorescent probe for mitochondrial thioredoxin [57]. The synthetic probe molecules Mito-Naph 7 react with thioredoxin to induce release of thiolate via intramolecular cyclization, resulting in fluorescent compounds.…”