Contamination of commercial preparations of xanthine oxidase by a Ca2+-dependent phospholipase A2

Abstract Using [1- 14 C]oleate-labelled autoclaved Escherichia coli as substrate, we demonstrate that many, but not all, commercial preparations of xanthine oxidase contain phospholipase A 2 activity as a contaminant. Phospholipase A 2 activity (64.3–545.6 nmol phospholipid hydrolyzed per min per mg protein) was optimal in the neutral to alkaline pH range, was Ca 2+ -dependent, and was unaffected by the addition of xanthine. Phospholipase A 2 activity was totally inhibited by 1.0 mM EDTA while radical production by xanthine plus xanthine oxidase was unaffected by EDTA. Even chromatographically purified xanthine oxidase (Sigma Grade III) contained substantial phospholipase A 2 activity (64.3 nmol/min per mg). Since the preparation of xanthine oxidase employs proteolytic digestion of milk or buttermilk by pancreatin, an extract of pancreas which is an organ rich in phospholipase A 2 activity, we speculate that the contaminant phospholipase A 2 is introduced by this treatment. Because xanthine oxidase is used extensively to study free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular phospholipase A 2 may have influenced previously published reports and such studies in the future should be interpreted with care.