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. 2009 Nov 23;285(5):3168–3180. doi: 10.1074/jbc.M109.056846

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

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FIGURE 1. — EPR spin trapping of generated from NQR in the presence of DEPMPO. A, the computer simulation (dashed line) superimposed on the experimental spectrum (solid line) obtained using NQR (0.1 mg/ml), DEPMPO (20 mm), diethylenetriaminopentaacetic acid (1 mm), and NADH (0.5 mm) in PBS. The experimental spectrum was recorded after signal averaging 3 scans at room temperature. B, the same as A, except that rotenone (25 μm) was added to the mixture before the reaction was initiated by NADH. C, the same as A, except that ubiquinone-1 (Q₁, 200 μm) was added to the mixture before the reaction was initiated by NADH. D, the same as C, except that rotenone (25 μm) was added to the mixture before the reaction was initiated by NADH. E, the same as A (or C) except that the NQR was omitted from the system. The spin quantitation for each spectrum was obtained by double integration of the simulation spectrum. The instrumental settings are described under “Experimental Procedures.” F, the same as C, except that DEPMPO was omitted from the system. G, the same as D, except that DEPMPO was omitted from the system. H, the same as G except that rotenone was replaced with piericidin A (250 nm) in the system. I, the EPR spectrum was recorded by the reaction mixture containing 2-(N,N-diethylamino)diazenolate 2-oxide (DEANO; 3 mm), Q₁H₂ (0.2 mm), and diethylenetriaminepentaacetic acid (1 mm) in 50 mm phosphate buffer, pH 7.4. The instrumental settings for F–I are: center field, 3525 G; sweep width, 20 G; power, 20 milliwatts; receiver gain, 2 × 10⁵; modulation amplitude, 1 G; time of conversion, 163.84 ms; time constant, 327.68 ms; and number of scans, 5.

Inline graphic — EPR spin trapping of generated from NQR in the presence of DEPMPO. A, the computer simulation (dashed line) superimposed on the experimental spectrum (solid line) obtained using NQR (0.1 mg/ml), DEPMPO (20 mm), diethylenetriaminopentaacetic acid (1 mm), and NADH (0.5 mm) in PBS. The experimental spectrum was recorded after signal averaging 3 scans at room temperature. B, the same as A, except that rotenone (25 μm) was added to the mixture before the reaction was initiated by NADH. C, the same as A, except that ubiquinone-1 (Q₁, 200 μm) was added to the mixture before the reaction was initiated by NADH. D, the same as C, except that rotenone (25 μm) was added to the mixture before the reaction was initiated by NADH. E, the same as A (or C) except that the NQR was omitted from the system. The spin quantitation for each spectrum was obtained by double integration of the simulation spectrum. The instrumental settings are described under “Experimental Procedures.” F, the same as C, except that DEPMPO was omitted from the system. G, the same as D, except that DEPMPO was omitted from the system. H, the same as G except that rotenone was replaced with piericidin A (250 nm) in the system. I, the EPR spectrum was recorded by the reaction mixture containing 2-(N,N-diethylamino)diazenolate 2-oxide (DEANO; 3 mm), Q₁H₂ (0.2 mm), and diethylenetriaminepentaacetic acid (1 mm) in 50 mm phosphate buffer, pH 7.4. The instrumental settings for F–I are: center field, 3525 G; sweep width, 20 G; power, 20 milliwatts; receiver gain, 2 × 10⁵; modulation amplitude, 1 G; time of conversion, 163.84 ms; time constant, 327.68 ms; and number of scans, 5.