Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system.
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.
Kim
Ju-A
JA
Center for Cell Signaling Research and Division of Molecular Life Sciences, Ewha Woman's University, Seoul, Korea.
Park
Sunjoo
S
Kim
Kangwha
K
Rhee
Sue Goo
SG
Kang
Sang Won
SW
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D010544:Peroxidases / Q000032:analysis* / Q000378:metabolism; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000032:analysis; D015003:Yeasts / Q000201:enzymology*
D005656:Fungal Proteins; D013879:Thioredoxins; D010544:Peroxidases; C504194:2-cys peroxiredoxin, human; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase