Abstract

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30. s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

Original languageEnglish (US)
Pages (from-to)517-521
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume439
Issue number4
DOIs
StatePublished - Oct 4 2013

Keywords

  • Glutathione
  • Green fluorescent protein (GFP)
  • Light-induced oxidation
  • Live cell imaging
  • Redox-sensitive probe

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Transient light-induced intracellular oxidation revealed by redox biosensor'. Together they form a unique fingerprint.

Cite this