Ostrich pancreatic phospholipase A2: Purification and biochemical characterization

Ostrich pancreatic phospholipase A2 (OPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 °C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA2, which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840 U/mg for purified OPLA2 was measured at optimal conditions (pH 8.2 and 37 °C) in the presence of 4 mM NaTDC and 10 mM CaCl2 using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C12-PC) monolayers. Maximal activity was measured at 5–8 mN m−1. The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA2 was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A2.