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 |
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Pages (from-to) | 36-40 |
Number of pages | 5 |
Journal | Analytical Biochemistry |
Volume | 393 |
Issue number | 1 |
DOIs | |
State | Published - 1 Oct 2009 |
Keywords
- 2-Cys peroxiredoxin
- Hyperoxidation
- Malachite green
- Phosphate assay
- Sulfiredoxin