Fig. 1.

HOBr generation by VPO1. (A) Verification of detection of HOBr by taurine-TMB oxidation assay. One hundred microliters of reaction containing 20 mM phosphate buffer, pH 7.4, 5 mM taurine, and HOCl or HOBr as indicated was carried out at 37 °C for 30 min. The reaction was stopped by adding catalase (25 µg/ml). The stopped reaction was then mixed with developing agent. After 5 min, absorbance at 650 nm was recorded. (B) pH-dependent HOBr generation by VPO1. One hundred microliters of reaction containing 20 mM phosphate buffer, pH as indicated, 100 µM KBr, 50 µM H₂O₂, 5 mM taurine, and 100 nM rVPO1, 20 nM MPO, or 100 nM bLPO was carried out at 37 °C for 30 min. The reaction was stopped by adding catalase (25 µg/ml). The stopped reaction was then mixed with developing agent. After 5 min, absorbance at 650 mm was recorded. (C) Dose-dependent HOBr generation by VPO1, compared with that by MPO. The experiments were carried out as in B at pH 7.4 containing 100 nM rVPO1 or 20 nM MPO, Concentrations of KBr and NaCl are indicated. (D) VPO1-dependent HOBr generation. The experiments were carried out as in B at pH 7.4, 100 µM Br⁻. The concentrations of rVPO1 and MPO are indicated. (E) Time course of rVPO1, MPO, and bLPO-mediated Br-tau formation. The experiments were carried out as in B at pH 7.4 with 100 µM Br⁻. Reactions started by adding hPx. Absorbance at 288 nm was continuously monitored. (F) Inhibition of HOBr generation. The experiments were carried out as in C with 100 µM Br⁻. In the reaction mixture, ABAH (200 µM), NaN₃ (1 mM), and catalase (25 µg/ml) were added, respectively. The data are from three independent experiments. Error bars, SD.