Fig. 2.

Assessment of NO formation from NDHP in the presence of XOR and different electron donors. NO formation was assessed in real-time using a chemiluminescence device and nitrogen gas as the NO carrier. The experimental conditions were: 1 mL reactional buffer (PBS, pH 7.4) containing XOR (0.05 U/mL), NDHP (1 mM) and the substrate A) NADH (1 mM), B) xanthine (20–500 µM), or D) 2,3-dihydroxybenzaldehyde (DBA, 500 µM). While only noise signals were detected in the presence of NDHP and XOR alone, adding NADH or DBA induced a distinct increase in nitric oxide formation. Conversely, increasing concentrations of xanthine induced no XOR-mediated NO generation from NDHP (Panel B). Similarly, the combination NDHP, XOR and xanthine (20 µM) induced no NO generation, but a later addition of nitrite (1 mM) to this mixture induced a distinct NO signal, which was inhibited by oxypurinol (Panel C). The tracings are representative of 4–6 independent experiments.