A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

Hojin Kim; Hyeryeon Kim; Sohyun Hong; Sue Goo Rhee; Woojin Jeong

doi:10.1016/j.ab.2009.06.030

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

Hojin Kim, Hyeryeon Kim, Sohyun Hong, Sue Goo Rhee, Woojin Jeong

Department of Life Science

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

6 Scopus citations

Abstract

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.

Original language	English
Pages (from-to)	36-40
Number of pages	5
Journal	Analytical Biochemistry
Volume	393
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2009.06.030
State	Published - 1 Oct 2009

Bibliographical note

Funding Information:
This work was supported by a grant from the Seoul R&BD program (10527), Bio R&D program grants to S.G.R. (M10642040001-07N4204-00110) and W.J. (M10642040002-07N4204-00210), a National Core Research Center program grant (R15-2006-020) through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science, and Technology, and funds from the Brain Korea 21 Scholars Program (to H.K. and S.H.).

Keywords

2-Cys peroxiredoxin
Hyperoxidation
Malachite green
Phosphate assay
Sulfiredoxin

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title = "A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement",

abstract = "2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H2O2). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg2+, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.",

keywords = "2-Cys peroxiredoxin, Hyperoxidation, Malachite green, Phosphate assay, Sulfiredoxin",

author = "Hojin Kim and Hyeryeon Kim and Sohyun Hong and Rhee, {Sue Goo} and Woojin Jeong",

note = "Funding Information: This work was supported by a grant from the Seoul R&BD program (10527), Bio R&D program grants to S.G.R. (M10642040001-07N4204-00110) and W.J. (M10642040002-07N4204-00210), a National Core Research Center program grant (R15-2006-020) through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science, and Technology, and funds from the Brain Korea 21 Scholars Program (to H.K. and S.H.).",

year = "2009",

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doi = "10.1016/j.ab.2009.06.030",

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journal = "Analytical Biochemistry",

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T1 - A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

AU - Kim, Hojin

AU - Kim, Hyeryeon

AU - Hong, Sohyun

AU - Rhee, Sue Goo

AU - Jeong, Woojin

N1 - Funding Information: This work was supported by a grant from the Seoul R&BD program (10527), Bio R&D program grants to S.G.R. (M10642040001-07N4204-00110) and W.J. (M10642040002-07N4204-00210), a National Core Research Center program grant (R15-2006-020) through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science, and Technology, and funds from the Brain Korea 21 Scholars Program (to H.K. and S.H.).

PY - 2009/10/1

Y1 - 2009/10/1

N2 - 2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H2O2). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg2+, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.

AB - 2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H2O2). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg2+, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.

KW - 2-Cys peroxiredoxin

KW - Hyperoxidation

KW - Malachite green

KW - Phosphate assay

KW - Sulfiredoxin

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A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

Abstract

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Keywords

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