Fig. 3.

Formation of the tyrosine oxidation product 3-nitroTyr on perlecan exposed to peroxynitrite: (A) effect of oxidant concentration, and (B) effect of reaction pH. Surface absorbed perlecan (10 nM) was treated in the absence (black bars) and presence (light grey bars) of NaHCO₃ (25 mM) for 20 min at 22 °C with (A) 0.1 M phosphate buffer, pH 7.5 containing 1 – 10 μM peroxynitrite (100 – 1000 molar excess of oxidant over protein) or dONOO (10 μM) or (B) 0.1 M phosphate buffer, pH 6 – 7.5 containing 10 μM peroxynitrite. The perlecan was then probed by ELISA using a mAb against 3-nitroTyr (HM11). Data are expressed as a % of the control ELISA signal obtained with untreated perlecan and are means ± SEM of values obtained from triplicate wells with n ≥ 3. The absolute absorbance (at 405 nm) for the controls with no perlecan were ∼ 0.055, and for native perlecan ∼ 0.09; those for the peroxynitrite-treated perlecan were up to 1.6 absorbance units. These data indicate that the level of 3-nitroTyr on the native perlecan was very low. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p < 0.05; a/A: different to native perlecan in the absence or presence of NaHCO₃ respectively, * effect of NaHCO₃.