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. 2010 May 10;285(28):21708–21723. doi: 10.1074/jbc.M110.117259

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — Dependence of the DEPMPO adducts on O₂. A, representative ESR spectra of reactions incubated aerobically (top) or anaerobically (bottom). The buffers, water, and NADPH were mixed and incubated for 5 min at 37 °C. Each reaction was started by adding TrxR (1.07 μm final concentration) and DEPMPO (14 mm final concentration). After 12 min at 37 °C, the reaction was loaded into an ESR flat cell and sealed, and spectra were acquired at room temperature. For the anaerobic experiments, the buffers and deionized water were preincubated in the anaerobic chamber for ≥24 h before use, and small volumes of NADPH, DEPMPO, and TrxR were pre-equilibrated for ≥4 h. The aerobic experiments were conducted identically, except all solutions and steps were handled under room air. Instrument settings were as described in the legend to Fig. 1. B, relative quantities of the HO^•, O, and carbon radical adducts of DEPMPO (mean ± S.D. for triplicate experiments) observed under aerobic versus anaerobic conditions. **, p < 0.01; ***, p < 0.001 versus the corresponding aerobic samples.

Inline graphic — Dependence of the DEPMPO adducts on O₂. A, representative ESR spectra of reactions incubated aerobically (top) or anaerobically (bottom). The buffers, water, and NADPH were mixed and incubated for 5 min at 37 °C. Each reaction was started by adding TrxR (1.07 μm final concentration) and DEPMPO (14 mm final concentration). After 12 min at 37 °C, the reaction was loaded into an ESR flat cell and sealed, and spectra were acquired at room temperature. For the anaerobic experiments, the buffers and deionized water were preincubated in the anaerobic chamber for ≥24 h before use, and small volumes of NADPH, DEPMPO, and TrxR were pre-equilibrated for ≥4 h. The aerobic experiments were conducted identically, except all solutions and steps were handled under room air. Instrument settings were as described in the legend to Fig. 1. B, relative quantities of the HO^•, O, and carbon radical adducts of DEPMPO (mean ± S.D. for triplicate experiments) observed under aerobic versus anaerobic conditions. **, p < 0.01; ***, p < 0.001 versus the corresponding aerobic samples.