Partial purification and characterization of arachidonate 12-lipoxygenase from rat lung

Incubation of rat lung cytosol with arachidonic acid produced 12-hydroxy-5,8,10,14-eicosatetraenoic acid as a major product, which was identified by gas chromatography-mass spectrometry. By ammonium sulfate fractionation and DEAE-cellulose chromatography the arachidonate 12-lipoxygenase was purified about 30-fold from the rat lung cytosol. The partially purified enzyme was mostly free of the glutathione peroxidase activity and transformed arachidonic acid to its 12-hydroperoxide. 5,8,11,14,17-Eicosapentaenoic acid was also an active substrate, and the oxygenation at C-12 was confirmed by mass spectrometry. A significant amount of 12-lipoxygenase activity was also found in the microsomes and other particulate fractions.