Abstract
This chapter presents the procedure for purification and assay of arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. All operations in purification were done at 0–4 °, crude extraction cells were sonicated 15 times for 30 sec in 60 ml of 50mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Further procedure steps are (1) acetone fractionation, (2) carboxymethyl-cellulose column chromatography, (3) gel filtration on sephadex G-150, and (4) hydroxyapatite column chromatography. The enzyme was eluted at approximately 350 mM buffer concentration. The active fractions were pooled and concentrated by ultrafiltration to about 1 ml. The activity of arachidonic acid-15-1ipoxygenase was assayed by measuring the sum of the radioactivities converted from [1-14C]arachidonic acid to 15-hydroperoxyeicosatetraenoic and its decomposition products. The radioactivity was determined in a scintillation counter. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1μmol of product in 2 min at 30 °.
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