Fig. 4.

XO activity facilitates oxyhemoglobin degradation. Oxyhemoglobin (17 μM) was incubated with XO (50 mU/mL) and xanthine (400 μM) for 20 min. Degradation of oxyhemoglobin and loss of iron was evaluated by eye A) and spectrophotometer B–C). D) EPR was used to follow the production of the ascorbate radical for assessing free iron and oxidant production. Oxyhemoglobin (17 μM) was incubated with ascorbate for 20 min prior to EPR measurement. E) Oxyhemoglobin (17 μM) was incubated with combinations of XO (50 mU/mL), and xanthine (400 μM) for 20 min and the relative signal intensity was normalized to ascorbate and oxyhemoglobin alone. F) DETA NONOate was used to create a continuous flow of 40 μM NO. Each reaction was added to the Sievers Nitric Oxide Analyzer sparger and NO consumption was quantified as G) area under the curve. ^$Values are mean ± SEM using a 1-way ANOVA with Dunnett multiple comparisons test. ^∼Values are mean ± SEM using a 1-way ANOVA with Sidak's multiple comparisons test. Min, minute; OxyHb, oxyhemoglobin; XO, xanthine oxidase; DTPA, Diethylenetriamine pentaacetate; EPR, electron paramagnetic resonance; DETA NONOate, (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; NO, nitric oxide.