A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement.
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.
Kim
Hojin
H
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.
Kim
Hyeryeon
H
Hong
Sohyun
S
Rhee
Sue Goo
SG
Jeong
Woojin
W
D003124:Colorimetry / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000032:analysis; D006801:Humans; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010710:Phosphates; D050862:Oxidoreductases Acting on Sulfur Group Donors; C504024:SRXN1 protein, human