Lipoxygenase activity assay of pLoxA

Lipoxygenase activity of pLoxA was assessed by formation of primary products of AA oxidation — 15-hydroperoxy-arachidonic acid (15-HpETE) or 1-SA-2-15-HpETE-PE. Briefly, 400 nM pLoxA was incubated with unilamellar liposomes of 1,2-dioleoyl-phosphatidylcholine (DOPC)/1-SA-2-AA-PE (1:1) or DOPC/AA (1:1) (100 nm) in the presence of 0.5 μM H₂O₂ and 100 μM DTPA (for transition metal chelation) in 20 mM HEPES saturated with oxygen, pH 7.4, at 37°C, in the absence or in the presence different concentrations of baicalein. At the end of incubation, AA and PE as well as their oxygenated products were extracted by the Folch procedure and analyzed by LC-MS using reverse phase column C30 as previously described (52 (link)). LC/ESI-MS/MS analysis of lipids was performed on a Dionex HPLC system (utilizing the Chromeleon software), consisting of a Dionex UltiMate 3000 mobile phase pump, equipped with an UltiMate 3000 degassing unit and UltiMate 3000 autosampler (sampler chamber temperature was set at 4^oC). The Dionex HPLC system was coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific).