FIG. 7.

Detection of tyrosine nitration. (A) Human PGHS-2 (5 μg) was treated with 0.1 μM 12-HpETE and 50 μM NO₂⁻ for 1 h, trypsin digested, and analyzed by electrospray ionization-Fourier transform mass spectrometry. All the detected peptides were labeled. The inserts represent the charge-state distribution of the different charge states of nitrated peptides 363–387. (B) For a comparison of the impact of NO₂⁻ versus peroxynitrite, PGHS-2 (5 μg) and 0.1 μM 12-HpETE were incubated either with NO₂⁻ (50 μM) or with the peroxynitrite donor Sin-1 (100 μM) in the presence or absence of substrate AA. (C) For an illustration of the role of peroxynitrite as an activator and inhibitor of PGHS-2, the enzyme (10 U/reaction) was treated with varying concentrations of the peroxynitrite-donor Sin-1 in the presence or absence of substrate AA (5 μM) for 30 min, and then ¹⁴C-AA was added for 5 min for activity determination. (D) Sin-1-treated PGHS-2 was subjected to the Western blot analysis and stained with an anti-3-NT antibody. Tyr, tyrosine.