Partial purification and characterization of a Ca²⁺-stimulated lipoxygenase from soybean seeds

Abstract

A highly purified Ca²⁺-stimulated lipoxygenase was isolated from the Hill variety of soybean seeds. Separation of Ca²⁺-stimulated lipoxygenase from lipoxygenase active in the absence of Ca²⁺ (lipoxygenase-1) was readily obtained using a DEAE-cellulose column. Sample size applied to the ion exchange column was found to be critical. Both enzymes were bound to the column, although some highly active Ca²⁺-stimulated lipoxygenase eluted with buffer in the presence of bound lipoxygenase-1. Ca²⁺-stimulated lipoxygenase bound to DAAE-cellulose required the use of a NaCl gradient for elution. Ca²⁺-stimulated lipoxygenase showed an apparent isoelectric point at pH 5.90 and optimum activity at pH 7.5 and at 1.1 mM calcium. Lipoxygenase-1 was inhibited over 95% in the presence of 60 μM methyl mercuric chloride, while Ca²⁺-stimulated lipoxygenase showed a maximum of only 20% inhibition under the same conditions.