Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems

Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9745-9. doi: 10.1073/pnas.152168599. Epub 2002 Jul 15.


To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with (35)S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to de-glutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cells, Cultured
  • Diamide / pharmacology
  • Glutathione / metabolism*
  • Glutathione Disulfide / metabolism
  • Humans
  • Insulin / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • T-Lymphocytes / metabolism
  • Thioredoxins / chemistry*
  • Thioredoxins / metabolism*
  • Trypsin


  • Insulin
  • Peptide Fragments
  • Diamide
  • Thioredoxins
  • Trypsin
  • Glutathione
  • Glutathione Disulfide