Irreversible hyperoxidation of peroxiredoxin 2 is caused by tert-butyl hydroperoxide in human red blood cells

Y I Ishida; M Takikawa; T Suzuki; M Nagahama; Y Ogasawara

doi:10.1016/j.fob.2014.10.003

Irreversible hyperoxidation of peroxiredoxin 2 is caused by tert-butyl hydroperoxide in human red blood cells

FEBS Open Bio. 2014 Oct 13:4:848-52. doi: 10.1016/j.fob.2014.10.003. eCollection 2014.

Authors

Y I Ishida¹, M Takikawa², T Suzuki², M Nagahama¹, Y Ogasawara²

Affiliations

¹ Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
² Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.

Abstract

Peroxiredoxin 2 (Prx2) is the third most abundant protein in red blood cells (RBCs). In this study, we have succeeded in implementing the rapid and simultaneous detection of the hyperoxidized (Prx2-SO2/3) and reduced (Prx2-SH) forms of Prx2 in human RBCs using reverse phase high-performance liquid chromatography. The detection of a peak corresponding to Prx2-SO2/3 was clearly observed following treatment of tert-butyl hydroperoxide (t-BHP), but not H2O2, and was found to be dose-dependent. The identity of the peak was confirmed as Prx2 by immunoblotting and mass spectrometry analysis. Our results suggest that t-BHP hyperoxidizes cysteine residues in Prx2 more readily than H2O2, and that accumulation of hyperoxidized Prx2 might reflect disruption of redox homeostasis in RBCs.

Keywords: Biomarker; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; HPLC, high-performance liquid chromatography; Hyperoxidation; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; Oxidative stress; PBS, phosphate-buffered saline; PMF, peptide mass fingerprinting; Peroxiredoxin 2; RBC, red blood cell; ROS, reactive oxygen species; Red blood cell; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TOF, time-of-flight; t-BHP, tert-butyl hydroperoxide; tert-Butyl hydroperoxide.