Abstract
The mechanism of prostaglandin synthase-dependent (bi)sulfite (hydrated sulfur dioxide) oxidation was investigated using an enzyme preparation derived from ram seminal vesicles. The horseradish peroxidase-catalyzed oxidation of (bi)sulfite was used as a model system. Incubation of (bi)sulfite with prostaglandin synthase and arachidonic acid, 15-hydroperoxyarachidonic acid, or H2O2 results in the formation of the reactive sulfur trioxide anion radical (SO3(-)). The horseradish peroxidase/H2O2 system also oxidizes (bi)sulfite to SO3(-). This free radical reacts with oxygen resulting in oxygen consumption by these incubations. The free radical was detected with the indirect electron spin resonance technique of spin trapping. The SO3(-) radical adduct formed by the reaction of SO3(-) with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, gives a nitroxide free radical with a nearly unique electron spin resonance spectrum (aH = 16.0 G and aN = 14.7 G). Using the spin-trapping technique, the SO3(-) could be detected even in incubations of guinea pig lung microsomes. When arachidonic acid-derived prostaglandin G2 was the source of hydroperoxide, formation of SO3(-) could be inhibited by indomethacin. When 15-hydroperoxyarachidonic acid or hydrogen peroxide was used to drive the enzymatic oxidation of (bi)sulfite, indomethacin had no effect. This hydroperoxidase activity was not nearly as heat-labile as the cyclo-oxygenase reaction which forms prostaglandin G2. Finally, the peroxidatic oxidation of (bi)sulfite may occur in vivo in competition with the mitochondrial sulfite oxidase, which oxidizes (bi)sulfite to sulfate without the formation of free radicals.
Highlights
The mechanism of prostaglandin synthase-dependentsulfite oxidation was investigated using an enzyme preparation derived from ram seminal vesicles
The peroxidatic oxidation ofsulfitemay occur in vivo in competition with the mitochondrial sulfite oxidase, which oxidizes(bi)sulfite to sulfate without the formation of free radicals
Despite the fact that the guinea pig lung microsomes contain approximately 200 times less prostaglandin synthase activity than ram seminal vesicles, the ESR spectra obtained from guinea pig lung microsomes indicate the formation of sulfur trioxide anion radical (SO): (Fig. 6 A ) .The signal was inhibited by indomethacin (Fig. 6s)and therewas some peroxidase activity, which could be inhibited by catalase (Fig. 6C)
Summary
Alase was obtained from Boehringer and arachidonic acid (99% pure) was obtained from Nu Chek Prep., Inc. (Elysian, MN). 5,5-DimethylI-pyrroline-N-oxide was obtained from Aldrich Chemical Co. and distilled before use. 15-Hydroperoxyarachidonicacid was prepared from arachidonic acid using soybean lipoxygenase (Sigma) according to published methods [21]. [l-'4C]Arachidonicacid was obtained from New England Nuclear. Enzyme Preparation the DMPO-SO; nitroxide adduct was predominant, i.e. other trapped radicals were in very low concentration and were. Microsomal prostaglandin synthase activity was determined by measuring arachidonic acid-dependent oxygen uptake using a Clark electrode. Free radicals sufiof guinea pig lung protein) were suspended in 0.5 ml of 100 mM ciently stable for observation by direct ESR are not usually sodium borate/boric acid buffer, pH 7.9, containing 1 mM diethyl- reactive enough to add across double bonds, and, enetriamine pentaacetic acid OG final concentrations were as follows:1m g / d of ram seminal vesicle microsomal protein, 1m~ Na2S03,500 p~ indomethacin, and. 410 p~ arachidonic acid or 10 p~ Hz& in sodium borate/boric acid buffer, pH 7.9, containing 1 m~ diethylenetriamine pentaacetic acid
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