FIGURE 8.

Effect of urate on LPO-mediated production of hypothiocyanite. The formation of hypothiocyanite by LPO was measured as oxidation of GSH to GSSG. A, hypothiocyanite was generated by the addition of 50 μm H₂O₂ to 10 nm LPO and 100 μm thiocyanate in the presence or absence of 250 μm urate in 10 mm phosphate buffer, pH 6.8. After a 10-min incubation at room temperature, 10 μg/ml catalase was added followed by N-ethylmaleimide. Data are means and standard deviations of five individual experiments. B, hypothiocyanite was produced over 1 h at 37 °C by incubation of 2 μg/ml glucose oxidase (GO) with 10 nm LPO, 100 μm thiocyanate, and 200 μm GSH in the presence or absence of 250 μm urate in 10 mm phosphate buffer, pH 6.8. Reactions were stopped by adding catalase (20 μg/ml), and then unreacted GSH was alkylated with 4 mm N-ethylmaleimide. Data are means and standard deviations for a representative experiment of 3–4 separate experiments. Data were analyzed by ANOVA using Holm-S̊ídák post hoc analysis. *, p < 0.05 for comparisons versus the complete reaction system.