Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Abstract

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM alpha-linolenic acid, 1.0 muM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA-H+O(2)].-CmDeltaP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C(2)H(5)-CH(CmDeltaP)-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C(2)H(5)-.CH-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity.