Activated eosinophils utilize plasma levels of bromide to covalently modify protein tyrosine residues. (a) Human eosinophils (1 × 10⁶/mL) were incubated at 37°C in Hanks’ balanced salt solution supplemented with DTPA (100 μM), BSA, 1 mg/mL, and bromide (100 μM NaBr). Eosinophils were activated with PMA (200 nM) and maintained in suspensions by intermittent inversion (Complete System). At the indicated times, eosinophils were removed by centrifugation. Supernatants were delipidated, desalted, and hydrolyzed, and the content of protein-bound 3-bromotyrosine and 3,5-dibromotyrosine was determined by stable isotope dilution GC-MS analysis. In a parallel set of experiments, superoxide production (in the absence of bromide and BSA) was determined as the superoxide dismutase-inhibitable reduction of ferricytochrome c. Data represent the mean ± SD of triplicate determinations (3-bromotyrosine and 3,5-dibromotyrosine) or a representative time course of O₂^•– production from an experiment performed at least four times. (b) The content of 3-bromotyrosine generated on target proteins after 2-hour incubation in the Complete System was determined by stable isotope dilution GC-MS analysis. Additions or deletions to the Complete System were as indicated. The final concentrations of additions were: catalase and heat inactivated catalase (hi Catalase), 10 μg/mL; NaN₃, 1 mM; 3-aminotriazole (Atz), 10 mM. Values are the mean ± SD of triplicate determinations. (c and d) Electron capture negative ion chemical ionization mass spectrum of n-propyl, per pentafluoroproprionyl derivatized (c) 3-bromotyrosine and (d) 3,5-dibromotyrosine recovered in amino acid hydrolysates of BSA exposed to activated eosinophils. (Insets) structures and proposed fragmentation pathways for derivatized (c) 3-bromotyrosine and (d) 3,5-dibromotyrosine.