Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.
The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.