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1.
Fig. 4

Fig. 4. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Effect of halides and pseudohalides on the formation of DMPO/SO3 radical. A reaction mixture containing 1 × 107 cells/ml, Na2SO3 (1 mM), and DMPO (100 mM) in PBS, pH 7.4. Na2SO3 (1 mM) was used for the remaining experiments. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell. The spectrum was attenuated in the presence of 100 mM NaCl (spectrum b), 100 μM NaBr (spectrum c), 100 μM NaSCN (spectrum d), and in the presence of the mixture of NaCl, NaBr, and NaSCN with the indicated concentrations (spectrum e).

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
2.
Fig. 6

Fig. 6. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Effect of Gd-DTPA as a line-broadening agent on the DMPO/SO3 radical adduct formation. The ESR spectrum of the DMPO/SO3 generated in a system of neutrophils (1 × 107 cells/ml), Na2SO3 (1 mM), and DMPO (100 mM) in PBS, pH 7.4. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell (spectrum a). (Spectrum b) Same as in spectrum a, but in the presence of 20 mM Gd-DTPA added prior to cell activation. (Spectrum c) Same as in spectrum b, but without neutrophils. (Spectrum d) Same as in spectrum a, but in the presence of 20 mM La-DTPA added prior to cell activation.

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
3.
Fig. 5

Fig. 5. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Effect of inhibitors and radical scavengers on the formation of DMPO/SO3 radical. Reaction mixture containing 1 × 107 cells/ml, Na2SO3 (1 mM), and DMPO (100 mM) in PBS, pH 7.4. Azide and ABAH were preincubated for 15 min before the addition of DMPO. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell. Na2SO3 (1 mM) was used for the remaining experiments. The spectrum was attenuated in the presence of NaN3 (500 μM) (spectrum b), ABAH (500 μM) (spectrum c), catalase (150 μg/ml) (spectrum d), and Cu, Zn-SOD (50 μM) (spectrum e).

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
4.
Fig. 3

Fig. 3. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Formation of radical adducts in reaction between sulfite (Na2SO3) and human neutrophils upon PMA activation in the presence of DMPO. (Spectrum a) Reaction mixture containing 1 × 107 cells/ml, Na2SO3 (1 mM), and DMPO (100 mM) in PBS, pH 7.4. After cell activation with PMA (500 ng/ml) followed by incubation at 37°C for 3 min, the mixture was placed into the flat cell. The dotted spectrum is the composite computer simulation of 26% DMPO/OOH (aN = 14.1 G, aHβ = 11.2 G, and aHγ = 1.24 G), 42% DMPO/OH (aN = 14.9 G and aHβ = 14.9 G), and 32% DMPO/SO3 (aN = 14.7 G and aHβ = 16.0 G) radical adducts. (Spectrum b) Same as in spectrum a without Na2SO3. (Spectrum c) Same as in spectrum a, except PMA was not added. (Spectrum d) Same as in spectrum a without Na2SO3 and PMA. (Spectrum e) Same as inspectrum a without neutrophils.

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
5.
Scheme 1

Scheme 1. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Sulfur dioxide, formed during the combustion of fossil fuels, is a major air pollutant [1]. It can be hydrated to (bi)sulfite in the lung upon contact with fluids lining the air passages (Scheme 1). Its two ionized forms in aqueous solution at physiological pH, (bi)sulfite (HSO3) and sulfite (SO32−) [2], are widely used in the food industry – predominantly as anti-browning agents, antioxidants and preservatives [3] - and as pharmaceutical ingredients [4]. It has also been reported that oral, topical or parenteral exposure to sulfites induces a wide range of adverse reactions in sensitive individuals and bronchoconstriction in asthmatic patients [4-6]. Until recently, there were only limited restrictions on the use of approved sulfiting agents in foods. These included a prohibition against their use in meats and a limitation of their concentrations in wines and raw shrimp to 350 ppm (5.5 mM) and 100 ppm (1.6 mM) SO2 equivalents, respectively [7].

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
6.

Fig. 1. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Reduction of MPO-compound II by sulfite. (A) Pseudo-first-order rate constants for reduction of MPO-compound I by (bi)sulfite. The second-order rate constant is calculated from the slope. The inset shows the time traces of the reaction followed at 430 nm using the sequential mixing mode. Final concentrations were 1.25 μM MPO and12.5 μM H2O2, and the concentration of (bi)sulfite for each time trace was (a) 12.5 μM, (b) 62.5 μM, (c) 125 μM, (d) 625 μM, and (e) 1.25 mM. (B) Spectral changes upon addition of 50 μM Na2SO3 to MPO-compound II. The resting MPO was recorded first (spectrum a). MPO-compound II was formed by mixing 400 nM ferric MPO with 300 nM homovanillic acid (HVA) and 50 μM H2O2 and waiting for 1 min (b). Spectrum c was taken 5 min after the addition of sulfite, and the resting enzyme was reformed after 30 min (spectrum d). (C) Pseudo-first-order rate constants for reduction of MPO-compound II by (bi)sulfite. The inset shows the time traces and fits of the reduction of compound II at pH 7.4 by Na2SO3. The concentration of (bi)sulfite for each time trace was (a) 10 μM, (b) 20 μM, (c) 50 μM, and (d) 100 μM.

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
7.
Fig. 2

Fig. 2. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

Formation of radical adducts in reaction between sulfite (Na2SO3) and myeloperoxidase (MPO)/hydrogen peroxide (H2O2) as a function of DMPO concentrations and time. (A) Reactions including Na2SO3 (1 mM) and MPO (1 μM) were initiated with H2O2 (100 μM) in 100 mM phosphate buffer (pH 7.4) in the presence of various concentrations of DMPO. After initiation with H2O2, the mixture was immediately placed into the flat cell. The concentration of the spin trap for each spectrum was (a) 100 mM, (b) 50 mM, (c) 20 mM, and (d) 6 mM. (B) Spectrum a is the same as spectrum d in Panel (A). Spectrum b is the composite simulation of 43% DMPO/OSO3, 33% DMPO/OH, and 24% DMPO/SO3. Spectrum c is the computer simulation of DMPO/OSO3 radical adduct (aN = 13.7 G, aHβ = 10.1 G, aHγ1 = 1.42 G, and aHγ2 = 0.75 G). Spectrum d is the simulation of DMPO/OH (aN = 14.9 G, aHβ = 14.9 G). Spectrum e is the simulation of DMPO/SO3 (aN = 14.6 G, aHβ = 16.2 G). (C) A reaction mixture containing Na2SO3 (1 mM), DMPO (6 mM), and MPO (1 μM) was initiated with H2O2 (100 μM) in 100 mM phosphate buffer (pH 7.4) and immediately placed into the flat cell (spectrum a). Spectra b, c, and d were detected after 3, 6, and 10 min, respectively. Spectrum e was detected immediately after the initiation, but in the presence of 100 mM DMSO. Spectrum f was detected immediately after the initiation, but in the presence of 100 mM HCOONa (the appearance of DMPO/CO2 radical adduct is marked with asterisks; aN = 15.8 G and aHβ = 18.7 G).

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.
8.
Scheme 2

Scheme 2. From: Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study.

In this study, we have shown that (bi)sulfite oxidation catalyzed by myeloperoxidase resulted in the formation of highly reactive sulfite-derived radicals. To our knowledge, this is the first report of sulfite oxidation by a mammalian peroxidase-H2O2 system resulting in detection of the highly reactive SO4•− radical. Our ESR experiments in the presence of 6 mM DMPO showed the formation of SO3, SO4•− and OH radical adducts, the latter building up with time (Fig. 2C). This is in agreement with previously published reports of spin trapping of SO4•− with DMPO, where nucleophilic substitution of the incipient leaving group in the initial radical adducts occurred via the reaction: DMPO/SO4•− + H2O → HSO4 + DMPO/OH [32, 34]. We were unable to detect the DMPO/OOSO3 radical adduct, probably because of the decomposition of DMPO/OOSO3 to DMPO/OH and O3SOOH [32]. The standard redox potential of SO4•− has been determined to be between 2.4 and 3.1 V (vs. NHE) [54, 55], which makes it as strong an oxidant as hydroxyl radical (E0 = 2.31 V) [56]. Although the enzyme-catalyzed oxidation of sulfites by other peroxidases has been shown to occur in lung microsomes [10], polymorphonuclear leukocytes [48], and lymphocytes from intestinal Peyer’s patches and mesenteric lymph nodes [57], there have been no previous reports in the literature of the myeloperoxidase metabolism of (bi)sulfite in neutrophils (Scheme 2).

Kalina Ranguelova, et al. Free Radic Biol Med. ;52(8):1264-1271.

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