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Anal Biochem. 2009 Oct 1;393(1):36-40. doi: 10.1016/j.ab.2009.06.030. Epub 2009 Jun 27.

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement.

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1
Department of Life Science, Division of Life and Pharmaceutical Sciences, and Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu Seoul 120-750, South Korea.

Abstract

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H(2)O(2)). 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, Mg(2+), 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.

PMID:
19563768
DOI:
10.1016/j.ab.2009.06.030
[Indexed for MEDLINE]

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