(A) study on lipoxygenase from soybean

Abstract

Soybean lipoxygenase (E.C.1.13.11.12) was purified by ammonium sulfate fractionation, affinity chromatography on linoleate-aminohexyl Sepharose 4B and DEAE-cellulose chromatography. Purification of lipoxygenase-2 through affinity chromatography at PH 6.8 resulted in 6-fold purification and yield of 91%. However, linoleate-aminoethyl Sepharose 4B did not retain a significant amount of lipoxygenase. Further purification was conducted through DEAE-cellulose chromatography with the factor of 4-fold, and the yield was 46 % with respect to lipoxygenase-2. Overall purification of lipoxygenase-2 was 32.4-fold, and yield was 28.5 %. The purfied lipoxygenase-2 was almost homogeneous, electrophoretically. The Km values of lipoxygenase-1 and -2 for linoleic acid were 0.15 mM and 0.39 mM, respectively. And the activation energies of the reactions catalyzed by lipoxygenase-1 and -2 were 16.2 kcal/mole and 39.6 kcal/mole, respectively. Molecular weight of lipoxygenase was determined from gel chromatography on Sephadex G-150 to be 102,000. When lipoxygenase-2 was subjected to be reacted with arachidonic acid, and subsequently reduced with sodium dithionite, a material from ether extracts of this reaction mixture seemed to have some of prostaglandin F_{2α}, which was identified through thin layer chromatography.