A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

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

Research output: Contribution to journalArticlepeer-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 languageEnglish
Pages (from-to)36-40
Number of pages5
JournalAnalytical Biochemistry
Volume393
Issue number1
DOIs
StatePublished - 1 Oct 2009

Keywords

  • 2-Cys peroxiredoxin
  • Hyperoxidation
  • Malachite green
  • Phosphate assay
  • Sulfiredoxin

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