Purification and characterization of a type-1 lipoxygenase from pea seeds

• Published in: Journal of agricultural and food chemistry 1982-01, Vol.30 (6), p.1157-1163
• Main Authors: Reynolds, Patricia A, Klein, Barbara P
• Format: Article
• Language: English
• Subjects: lipoxygenase; Pisum
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf00114a039

A type-1 lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) was purified from pea seeds by a combination of ammonium sulfate fractionation, gel filtration, ion-exchange chromatography, and preparative isoelectric focusing in a granulated gel. Lipoxygenase-1 was very unstable, especially at pH values below 6, and extensive loss of enzyme activity occurred during preparative isoelectric focusing. Partially purified lipoxygenase-1 focused into enzyme-active bands at pH 4.05 and 4.20. This preparation effectively catalyzed the oxidation of linoleate, linolenate, methyl linoleate, and trilinolein substrates but exhibited much lower activity than type-2 pea lipoxygenases. Highest activity occurred with linoleic acid, with maximum activity in the 9.0-10.0 range and an apparent K sub(m) of 0.20 mM at pH 9.0. Apparent molecular weights of 64,000 and 65,000 were obtained by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isoenzyme with pI = 4.05, respectively. Like soybean lipoxygenase-1, pea lipoxygenase-1 was not as effective in carotene cooxidation as the type-2 enzymes, and production of 280 nm absorbing compounds occurred only after the system became anaerobic. Lipoxygenase-1 was ineffective in bleaching chlorophyll.