Abstract
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.
Highlights
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
Free radicals have been demonstrated to be produced by enzymatic initiation of the oxidation of bisulfite by prostaglandin H synthase [9] and horseradish peroxidase [10, 11], with formation of the 1⁄7SO3Ϫ anion radical as follows: Native EPO ϩ H2O2 3 EPO-compound I ϩ H2O REACTION 3 where k ϭ 4.3 ϫ 107 MϪ1 sϪ1 [12], and EPO-compound I ϩ SO32Ϫ 3 EPO-compound II ϩ 1⁄7SO3Ϫ EPO-compound II ϩ SO32Ϫ 3 Native EPO ϩ 1⁄7SO3Ϫ REACTIONS 4 AND 5
The proposed mechanism of enzymatic oxidation of bisulfite to the 1⁄7SO3Ϫ radical by the eosinophil peroxidase/H2O2 system proceeds in two sequential, one-electron reduction reactions of compounds I and II by sulfite, similar to the oxidation of bisulfite by horseradish peroxidase and prostaglandin H synthase [9, 11, 15, 36]
Summary
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. When we examined the DMPO concentration dependence of oxygen consumption experiments using 2 mM bisulfite, 100 M H2O2, and 50 nM EPO as the initiator, the prior addition of 100 mM DMPO (the same amount used for the ESR data) almost completely prevented oxygen uptake (Fig. 3B, trace a)
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