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

Ju A. Kim, Sunjoo Park, Kangwha Kim, Sue Goo Rhee, Sang Won Kang

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

Abstract

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 2O2-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 μM, 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.

Original languageEnglish
Pages (from-to)216-223
Number of pages8
JournalAnalytical Biochemistry
Volume338
Issue number2
DOIs
StatePublished - 15 Mar 2005

Keywords

  • 2-Cys peroxiredoxin
  • Thioredoxin peroxidase
  • Yeast thioredoxin
  • Yeast thioredoxin reductase

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