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

doi:10.1016/j.ab.2004.12.008

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

Department of Life Science

Research output: Contribution to journal › Article › peer-review

57 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 language	English
Pages (from-to)	216-223
Number of pages	8
Journal	Analytical Biochemistry
Volume	338
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2004.12.008
State	Published - 15 Mar 2005

Bibliographical note

Funding Information:
We thank Dr. Jaesang Kim for critically reading the manuscript. We also thank Dr. Ho Jong Kwon for sharing the instrument and members of Labfrontier Co (Korea) for valuable technical support. This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Center for Cell Signaling Research and a Junior Faculty Research Grant at Ewha Womans University. S.J.P. was partially supported by the SK-Ewha research fund and J.K. was a recipient of Brain Korea 21 grant.

Keywords

2-Cys peroxiredoxin
Thioredoxin peroxidase
Yeast thioredoxin
Yeast thioredoxin reductase

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10.1016/j.ab.2004.12.008

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title = "Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system",

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.",

keywords = "2-Cys peroxiredoxin, Thioredoxin peroxidase, Yeast thioredoxin, Yeast thioredoxin reductase",

author = "Kim, {Ju A.} and Sunjoo Park and Kangwha Kim and Rhee, {Sue Goo} and Kang, {Sang Won}",

note = "Funding Information: We thank Dr. Jaesang Kim for critically reading the manuscript. We also thank Dr. Ho Jong Kwon for sharing the instrument and members of Labfrontier Co (Korea) for valuable technical support. This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Center for Cell Signaling Research and a Junior Faculty Research Grant at Ewha Womans University. S.J.P. was partially supported by the SK-Ewha research fund and J.K. was a recipient of Brain Korea 21 grant.",

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AU - Kim, Ju A.

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AU - Rhee, Sue Goo

AU - Kang, Sang Won

N1 - Funding Information: We thank Dr. Jaesang Kim for critically reading the manuscript. We also thank Dr. Ho Jong Kwon for sharing the instrument and members of Labfrontier Co (Korea) for valuable technical support. This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Center for Cell Signaling Research and a Junior Faculty Research Grant at Ewha Womans University. S.J.P. was partially supported by the SK-Ewha research fund and J.K. was a recipient of Brain Korea 21 grant.

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N2 - 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.

AB - 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.

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JF - Analytical Biochemistry

IS - 2

ER -

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

Abstract

Bibliographical note

Keywords

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