Eosinophil peroxidase (EPO) is an abundant
heme protein in eosinophils that catalyzes the formation of cytotoxic
oxidants implicated in
asthma, allergic inflammatory disorders, and
cancer. It is known that some
proteins with
peroxidase activity (
horseradish peroxidase and
prostaglandin hydroperoxidase) can catalyze oxidation of
bisulfite (hydrated
sulfur dioxide), leading to the formation of
sulfur trioxide anion radical ((.)SO(3)(-)). This
free radical further reacts with
oxygen to form
peroxymonosulfate anion radical ((-)O(3)SOO(.)) and the very reactive
sulfate anion radical (SO(4)()), which is nearly as strong an
oxidant as the
hydroxyl radical. However, the ability of EPO to generate reactive
sulfur radicals has not yet been reported. Here we demonstrate that
eosinophil peroxidase/H(2)O(2) is able to oxidize
bisulfite, ultimately forming the
sulfate anion radical (SO(4)()), and that these reactive intermediates can oxidize target
proteins to
protein radicals, thereby initiating
protein oxidation. We used immuno-spin trapping and confocal microscopy to study
protein oxidation by EPO/H(2)O(2) in the presence of
bisulfite in a pure enzymatic system and in human promyelocytic
leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-
oxide (DMPO) detected the presence of DMPO covalently attached to the
proteins resulting from the DMPO trapping of
protein free radicals. We found that
sulfite oxidation mediated by EPO/H(2)O(2) induced the formation of radical-derived DMPO spin-trapped
human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in
bisulfite-exacerbated eosinophilic inflammatory disorders.