New insights into the detection of sulfur trioxide anion radical by spin trapping: radical trapping versus nucleophilic addition

Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 05/2009; 47(2):128-34. DOI: 10.1016/j.freeradbiomed.2009.04.006
Source: PubMed


It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) reacts with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) via a nonradical, nucleophilic reaction, and further proposed that the radical adduct (DMPO/()SO(3)(-)) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical (()SO(3)(-)). Here, the one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase/H(2)O(2) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the case of ESR experiments, the signal of the DMPO/()SO(3)(-) radical adduct was detected, and the initial rate of its formation was calculated. Support for the radical pathway via ()SO(3)(-) was obtained from the stoichiometry between the amount of consumed molecular oxygen and the amount of (bi)sulfite oxidized to sulfate (SO(4)(2-)). When DMPO was incubated with (bi)sulfite, oxygen consumption was completely inhibited owing to the efficiency of DMPO trapping. In the absence of DMPO, the initial rate of oxygen and H(2)O(2) consumption was determined to be half of the initial rate of DMPO/()SO(3)(-) radical adduct formation as determined by ESR, demonstrating that DMPO forms the radical adduct by trapping the ()SO(3)(-) exclusively. We conclude that DMPO is not susceptible to artifacts arising from nonradical chemistry (nucleophilic addition) except when both (bi)sulfite and DMPO concentrations are at nonphysiological levels of at least 0.1 M and the incubations are for longer times.

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Available from: Ronald P Mason
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    • ") and HRP (1.8 ± 0.06 × 10 2 M −1 s −1 [30]). Although the calculated rates show that sulfite is a relatively poor myeloperoxidase substrate, it has been demonstrated that the sulfite radical chain chemistry via Eqns (4)-(6) can be initiated by only 1.4 × 10 −13 M • SO 3 − [31]. "
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    ABSTRACT: The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((•)SO(3)(-)) and sulfate (SO(4)(•-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(•-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.
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    • "•– / SO 4 2– = 2.43 V)—can attack target proteins (e.g., HSA in plasma) (Neta et al. 1988; Steele and Appelman 1982) (Figure 1). Previous work on the oxidation of (bi)sulfite by the HRP–H 2 O 2 system and ESR spin­ trapping experiments showed that there is a strong competition between the spin trap DMPO and oxygen for • SO 3 – (Ranguelova and Mason 2009). In fact, in the latter system, the formation of the oxygen­derived radicals – O 3 SOO • and SO 4 •– was almost prevented by high DMPO concentrations (100 mM) (Figure 3B), and a decrease of the spin­trap concentration to ≤ 3 mM was required to trap protein radicals formed by – O 3 SOO • and SO 4 •– (Mottley and Mason 1988). "
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