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.
Bibliographical noteFunding 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.).
- 2-Cys peroxiredoxin
- Malachite green
- Phosphate assay