Purification of bovine pancreatic phospholipase A 2 by an affinity ultrafiltration technique

Abstract

Phospholipase A 2 (PLA 2; EC 3.1.1.4) is a lipolytic enzyme that hydrolyze phosphoglycerides at the acyl ester bond at the sn-2-position into the corresponding lysophospholipid and fatty acid. Mammalian PLA 2 enzymes are subdivided into three groups: low molecular weight Ca 2+-dependent enzymes (sPLA 2), high molecular weight Ca 2+-dependent enzymes (cPLA 2) and the Ca 2+-independent isoforms (iPLA 2). The object of this study is to discover the synthesis of an affinity complex which could be used in an ultrafiltration system for the biospecific affinity isolation of phospholipase A 2 from mixtures containing other proteins. For this purpose, first, the development of a macromolecular water soluble resin was attempted, based on chitosan bearing phosphatidylethanolamine, a phospholipase A 2 substrate. Then, together with ultrafiltration techniques, the macroligand was employed to purify phospholipase A 2 from bovine pancreas. The enzyme was purified 79-fold and had a high activity yield 76.3%. The enzymatic properties obtained and showed nearly similarity in terms of optimum temperature and pH, molecular weight, Ca 2+-dependence with bovine and other mammalian pancreatic PLA 2's.