Arachidonate 12-lipoxygenase of porcine leukocyte with activity for 5-hydroxyeicosatetraenoic acid.

When arachidonic acid was incubated with porcine polymorphonuclear leukocytes, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was produced as a major product. The production of this compound was not accounted for by contaminating platelets known to contain arachidonate 12-lipoxygenase. The enzyme was found predominantly in a cytosol fraction of leukocytes, and the cytosolic enzyme was purified about 30-fold by ammonium sulfate fractionation and DEAE-Sephadex column chromatography. The partially purified enzyme transformed arachidonic acid to a compound which was identified as 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid as substrate was about 40% as active as arachidonic acid, and the reaction was inhibited by the addition of arachidonic acid. The reaction product which was further reduced by sodium borohydride was indistinguishable from authentic 5S, 12S-dihydroxy-(E,Z,E,Z)-6,8,10,14-eicosatetraenoic acid.