Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

Anal Biochem. 2005 Mar 15;338(2):216-23. doi: 10.1016/j.ab.2004.12.008.


2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Fungal Proteins / analysis
  • Fungal Proteins / genetics
  • Humans
  • Peroxidases / analysis*
  • Peroxidases / metabolism
  • Peroxiredoxins
  • Thioredoxin-Disulfide Reductase / analysis
  • Thioredoxin-Disulfide Reductase / genetics
  • Thioredoxin-Disulfide Reductase / metabolism*
  • Thioredoxins / analysis
  • Yeasts / enzymology*


  • Fungal Proteins
  • Thioredoxins
  • Peroxidases
  • 2-cys peroxiredoxin, human
  • Peroxiredoxins
  • Thioredoxin-Disulfide Reductase