Irreversible hyperoxidation of peroxiredoxin 2 is caused by tert-butyl hydroperoxide in human red blood cells.
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.
Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
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Ishida
Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
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Takikawa
Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
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Suzuki
Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
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Nagahama
Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
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Ogasawara
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