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

Ishida YI, Takikawa M, Suzuki T, Nagahama M, Ogasawara Y - FEBS Open Bio (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular 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.

No MeSH data available.


Chromatograms of human RBC lysate as analyzed by reverse phase chromatography and Western blot analysis. Reverse phase HPLC was performed using an YMC-pack PROTEIN-RP column (4.6 × 250 mm) as described in Section 2. The separated proteins were collected in 1.0 ml fractions and concentrated using a centrifugation concentrator system (Sakuma, Tokyo, Japan) for further analysis. After concentration, all fractions (3–50 min) were subjected to Western blot analysis with anti-Prx2 antibody after 12% SDS–PAGE and transfer to a PVDF membrane. RBC lysates were prepared after treatment with 500 μM H2O2 (B), t-BHP (C) or without peroxide (A). A typical result from the other three samples providing similar results is presented.
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f0005: Chromatograms of human RBC lysate as analyzed by reverse phase chromatography and Western blot analysis. Reverse phase HPLC was performed using an YMC-pack PROTEIN-RP column (4.6 × 250 mm) as described in Section 2. The separated proteins were collected in 1.0 ml fractions and concentrated using a centrifugation concentrator system (Sakuma, Tokyo, Japan) for further analysis. After concentration, all fractions (3–50 min) were subjected to Western blot analysis with anti-Prx2 antibody after 12% SDS–PAGE and transfer to a PVDF membrane. RBC lysates were prepared after treatment with 500 μM H2O2 (B), t-BHP (C) or without peroxide (A). A typical result from the other three samples providing similar results is presented.

Mentions: First, we investigated the separation conditions for Prx2 in RBC lysates using reverse-phase columns for protein separation. The eluted fractions were subjected to SDS–PAGE, followed by Western blotting detection with anti-Prx2 antibodies. Fig. 1A shows a chromatogram of lysates from native RBCs and the separation pattern of Prx2. Using the gradient program that we established in this study, Prx2 was detected at a retention time of 42 min (Peak-a, Fig. 1A). Additionally, a minor band was observed at a retention time of 46 min. We next examined the effect of oxidative stress on Prx2 elution using the commonly employed oxidant H2O2. Compared to control (Fig. 1A), changes in the chromatogram and separation pattern of Prx2 were not prominent in H2O2-treated RBCs (Fig. 1B), although the low intensity Prx2 band at a retention time of 46 min tended to increase. Following pretreatment with inhibitors (10 mM 3-AT for catalase, 7 mM succinate for GPx1) before exposure of RBC suspensions to 500 μM H2O2, no significant hyperoxidation of Prx2 was observed (data not shown).


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

Ishida YI, Takikawa M, Suzuki T, Nagahama M, Ogasawara Y - FEBS Open Bio (2014)

Chromatograms of human RBC lysate as analyzed by reverse phase chromatography and Western blot analysis. Reverse phase HPLC was performed using an YMC-pack PROTEIN-RP column (4.6 × 250 mm) as described in Section 2. The separated proteins were collected in 1.0 ml fractions and concentrated using a centrifugation concentrator system (Sakuma, Tokyo, Japan) for further analysis. After concentration, all fractions (3–50 min) were subjected to Western blot analysis with anti-Prx2 antibody after 12% SDS–PAGE and transfer to a PVDF membrane. RBC lysates were prepared after treatment with 500 μM H2O2 (B), t-BHP (C) or without peroxide (A). A typical result from the other three samples providing similar results is presented.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215121&req=5

f0005: Chromatograms of human RBC lysate as analyzed by reverse phase chromatography and Western blot analysis. Reverse phase HPLC was performed using an YMC-pack PROTEIN-RP column (4.6 × 250 mm) as described in Section 2. The separated proteins were collected in 1.0 ml fractions and concentrated using a centrifugation concentrator system (Sakuma, Tokyo, Japan) for further analysis. After concentration, all fractions (3–50 min) were subjected to Western blot analysis with anti-Prx2 antibody after 12% SDS–PAGE and transfer to a PVDF membrane. RBC lysates were prepared after treatment with 500 μM H2O2 (B), t-BHP (C) or without peroxide (A). A typical result from the other three samples providing similar results is presented.
Mentions: First, we investigated the separation conditions for Prx2 in RBC lysates using reverse-phase columns for protein separation. The eluted fractions were subjected to SDS–PAGE, followed by Western blotting detection with anti-Prx2 antibodies. Fig. 1A shows a chromatogram of lysates from native RBCs and the separation pattern of Prx2. Using the gradient program that we established in this study, Prx2 was detected at a retention time of 42 min (Peak-a, Fig. 1A). Additionally, a minor band was observed at a retention time of 46 min. We next examined the effect of oxidative stress on Prx2 elution using the commonly employed oxidant H2O2. Compared to control (Fig. 1A), changes in the chromatogram and separation pattern of Prx2 were not prominent in H2O2-treated RBCs (Fig. 1B), although the low intensity Prx2 band at a retention time of 46 min tended to increase. Following pretreatment with inhibitors (10 mM 3-AT for catalase, 7 mM succinate for GPx1) before exposure of RBC suspensions to 500 μM H2O2, no significant hyperoxidation of Prx2 was observed (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular 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.

No MeSH data available.