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1.
FIGURE 8.

FIGURE 8. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Proposed reaction pathways in the oxidation of CBA to COH.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
2.
FIGURE 1.

FIGURE 1. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Scheme showing the conversion of CBA and CBE into the fluorescent product, COH.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
3.
FIGURE 3.

FIGURE 3. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Stoichiometry of the reaction between CBA and ONOO. The concentrations of CBA and COH calculated from HPLC data were obtained from incubations containing CBA (100 μm), 20 μm DTPA, and ONOO (0–200 μm) in phosphate buffer (0.1 m, pH 7.4). Error bars indicate S.D.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
4.
FIGURE 4.

FIGURE 4. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Kinetics of the reaction between CBA and ONOO. A–C, stopped-flow kinetic traces obtained after mixing CBA (20 μm) and ONOO (10 μm) in phosphate buffer (0.25 m, pH 7.4), using the absorption detector at 286 nm (A) and 370 nm (B) or using the fluorescence detector (C, excitation at 332 nm, emission at > 400 nm). a.u., arbitrary units. D, the dependence of the pseudo first-order rate constant of COH formation (as detected by fluorescence) on the CBA concentration; [ONOO] = 0.1 μm.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
5.
FIGURE 5.

FIGURE 5. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Detection of ONOO formed from co-generated superoxide and nitric oxide fluxes. The fluorescence intensity (excitation at 332 nm, emission at 450 nm) of the reaction mixtures consisting of CBA (100 μm) with or without X/XO (O2˙̄ flux: 2 μm/min) and PAPA-NONOate (NO flux: 2 μm/min) was measured over a period of 30 min. The reaction mixtures contained catalase (0.1 kU/ml) and DTPA (10 μm) in phosphate buffer (100 mm, pH 7.4). a.u., arbitrary units.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
6.
FIGURE 2.

FIGURE 2. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Fluorescent spectral changes during the reaction between CBA and peroxynitrite. Fluorescence (Fl) spectra (excitation/emission) were obtained from incubations containing CBA (50 μm) and DTPA (50 μm) in phosphate buffer (0.45 m, pH 7.4) after the addition of various amounts of ONOO (0–2 μm). Emission spectra were collected using excitation at 332 nm; excitation spectra were collected by monitoring the emission intensity at 451 nm. Insets, HPLC traces recorded from the incubation mixtures containing CBA (100 μm), before (control, top trace) and after (bottom trace) the addition of ONOO (20 μm). The left panel represents the signal detected using the absorption (Abs) detector set at 300 nm; the right panel represents the signal detected using the fluorescence detector with the excitation set at 332 nm and emission at 470 nm. HPLC peaks at 9.5 and 10.7 min correspond to CBA and COH, respectively. a.u., arbitrary units.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
7.
FIGURE 6.

FIGURE 6. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Detection of ONOO formed from co-generated superoxide and nitric oxide fluxes; effects of superoxide dismutase and catalase (CAT). A, the time course of the fluorescence intensity increase (excitation at 332 nm, emission at 450 nm) measured in reaction mixtures containing CBA (100 μm) and DTPA (10 μm) in phosphate buffer (100 mm, pH 7.4) with or without X (200 μm), XO (O2˙̄ flux: 1.2 μm/min), and PAPA-NONOate (NO flux: 2.7 μm/min). B, effects of SOD (1 mg/ml) and catalase (100 units/ml) on the total change in the fluorescence intensity after 30 min of incubation. Error bars indicate S.D. a.u., arbitrary units. C, COH concentrations in the reaction mixtures measured by HPLC after a 30-min incubation under similar conditions but with NO and O2˙̄ fluxes of 2.2 μm/min and SOD concentration of 0.2 mg/ml. Open and solid symbols/bars represent the values obtained from mixtures in the absence and presence of PAPA-NONOate, respectively.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.
8.
FIGURE 7.

FIGURE 7. From: Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide.

Detection of ONOO formed from co-generated superoxide and nitric oxide fluxes; effects of varying NO/O2˙̄ ratios on product formation. A, the time course of the increase in the fluorescence intensity (excitation at 332 nm, emission at 450 nm) obtained at room temperature from the incubation mixtures containing CBA (100 μm), 10 μm DTPA, 100 units/ml catalase, xanthine (200 μm), XO (O2˙̄ flux: 2 μm/min), and different concentrations of PAPA-NONOate (NO flux: 0–12 μm/min) in phosphate buffer (0.1 m and pH 7.4). NO fluxes (μm/min) are: a, 0; b, 0.75; c, 1.5; d, 2.25; e, 3; f, 6; g, 9; h, 12. a.u., arbitrary units. B, rate of increase in the fluorescence intensity versus NO flux. Error bars indicate S.D. C, HPLC measurements of COH and uric acid formed after 30 min of incubation under similar conditions but with an O2˙̄ flux of 2.2 μm/min. D, the concentration of COH after correcting for the inactivation of the enzyme, XO. Note that in panels C and D, the x axis scale is not linear.

Jacek Zielonka, et al. J Biol Chem. 2010 May 7;285(19):14210-14216.

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