Limits...
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.


Oxidative status of Prx2 in human RBCs following t-BHP treatment. An RBC lysate was prepared after treatment with 500 μM t-BHP. After concentration, each fraction was subjected to Western blot analysis with anti-Prx2 antibody (A; upper image) and reprobed with anti-Prx-SO2/3 antibody (B; lower image). Peaks b and c are indicated on the chromatogram. A typical result from two other samples exhibiting similar responses is presented.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215121&req=5

f0025: Oxidative status of Prx2 in human RBCs following t-BHP treatment. An RBC lysate was prepared after treatment with 500 μM t-BHP. After concentration, each fraction was subjected to Western blot analysis with anti-Prx2 antibody (A; upper image) and reprobed with anti-Prx-SO2/3 antibody (B; lower image). Peaks b and c are indicated on the chromatogram. A typical result from two other samples exhibiting similar responses is presented.

Mentions: To clarify the oxidative status of Prx2, two-dimensional (2D) electrophoresis of the t-BHP-treated sample was performed, followed by immunoblot detection of Prx2. The results showed two spots of equal molecular weight with pI values of 5.8 and 5.4; the more acidic spot (pI 5.4) is more clearly observed in the t-BHP-treated sample compared to the control sample (Fig. 4). Furthermore, the two peaks obtained from the oxidized samples by reverse phase HPLC were also examined by immunoblotting. While similar band intensities (23 kDa) were observed for both peaks detected with the anti-Prx2 antibody, anti-PrxSO2/3 immunoreactivity was markedly increased in Peak-c compared to Peak-b (Fig. 5). These results indicated that the native and hyperoxidized forms of Prx2 were separated by our HPLC system.


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)

Oxidative status of Prx2 in human RBCs following t-BHP treatment. An RBC lysate was prepared after treatment with 500 μM t-BHP. After concentration, each fraction was subjected to Western blot analysis with anti-Prx2 antibody (A; upper image) and reprobed with anti-Prx-SO2/3 antibody (B; lower image). Peaks b and c are indicated on the chromatogram. A typical result from two other samples exhibiting similar responses is presented.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0025: Oxidative status of Prx2 in human RBCs following t-BHP treatment. An RBC lysate was prepared after treatment with 500 μM t-BHP. After concentration, each fraction was subjected to Western blot analysis with anti-Prx2 antibody (A; upper image) and reprobed with anti-Prx-SO2/3 antibody (B; lower image). Peaks b and c are indicated on the chromatogram. A typical result from two other samples exhibiting similar responses is presented.
Mentions: To clarify the oxidative status of Prx2, two-dimensional (2D) electrophoresis of the t-BHP-treated sample was performed, followed by immunoblot detection of Prx2. The results showed two spots of equal molecular weight with pI values of 5.8 and 5.4; the more acidic spot (pI 5.4) is more clearly observed in the t-BHP-treated sample compared to the control sample (Fig. 4). Furthermore, the two peaks obtained from the oxidized samples by reverse phase HPLC were also examined by immunoblotting. While similar band intensities (23 kDa) were observed for both peaks detected with the anti-Prx2 antibody, anti-PrxSO2/3 immunoreactivity was markedly increased in Peak-c compared to Peak-b (Fig. 5). These results indicated that the native and hyperoxidized forms of Prx2 were separated by our HPLC system.

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.