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Related Experiment Video

Updated: May 18, 2026

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo
12:06

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Published on: November 5, 2013

Mitochondrial thioredoxin-responding off-on fluorescent probe.

Min Hee Lee1, Ji Hye Han, Jae-Hong Lee

  • 1Department of Chemistry, Korea University, Seoul, 136-701, Korea.

Journal of the American Chemical Society
|September 29, 2012
PubMed
Summary

We developed Mito-Naph, a novel fluorescent probe, to accurately measure mitochondrial thioredoxin (Trx) activity in cells. This probe enables sensitive detection of Trx at nanomolar concentrations, offering insights into cellular redox balance.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Chemical Biology

Background:

  • Mitochondrial thioredoxin (Trx) plays a crucial role in cellular redox homeostasis.
  • Accurate visualization and quantification of Trx activity are essential for understanding its biological functions.
  • Existing methods for detecting Trx activity have limitations in sensitivity and specificity.

Purpose of the Study:

  • To synthesize and characterize a novel fluorescent probe, Mito-Naph, for visualizing mitochondrial Trx activity.
  • To establish Mito-Naph as a sensitive and selective tool for detecting Trx in vitro and in vivo.
  • To investigate the kinetics and specificity of Mito-Naph's reaction with Trx.

Main Methods:

  • Synthesis of the Mito-Naph fluorescent probe.
  • Fluorometric assays to measure Trx activity and probe kinetics.
  • Confocal microscopy to visualize mitochondrial Trx activity in living cells.
  • Inhibition studies using PX-12, a Trx inhibitor.
  • Immunoprecipitation to deplete Trx and assess probe response.

Main Results:

  • Mito-Naph exhibits an "off-on" fluorescence response upon disulfide cleavage by Trx.
  • The probe detects Trx activity at nanomolar concentrations (down to 50 nM).
  • Mito-Naph demonstrates high specificity for Trx over other biological thiols, with a reaction rate approximately 5000 times faster than for GSH.
  • Inhibition and depletion studies confirm the probe's selective reaction with Trx in living cells and cell extracts.

Conclusions:

  • Mito-Naph is a highly sensitive and selective fluorescent probe for visualizing and quantifying mitochondrial Trx activity.
  • The probe's characteristics make it a valuable tool for studying cellular redox processes and the role of Trx in various biological contexts.
  • Mito-Naph facilitates the study of Trx in both in vitro and in vivo settings with high precision.