Purification and activity of two phospholipase enzymes from Naja nigricolis nigricolis reinhardt venom

Two phospholipase enzymes NN1 and NN2 were purified from the venom of Naja nigricolis nigricolis Reinhardt to apparent homogeneity. NN1 was purified by a two‐step anion‐exchange chromatography on DEAE‐cellulose column while NN2 was purified by a combination of anion‐exchange chromatography and gel filteration on Sephadex G‐150. The enzyme NN1 moved homogenously on acrylamide gel as a monomer with a molecular weight of 65 kDa while NN2 was a dimer of 71 kDa. Both enzymes were clearly separated. Both enzymes hydrolyzed L ‐α‐phosphatidyl choline with activities of 345.5 for NN1 and 727.8 μmol min −1 mg −1 for NN2. The dimeric 71‐kDa enzyme has a higher haemolytic and anticoagulant activity than the monomeric 65‐kDa enzyme. It is apparent that the dimeric enzyme has a more pronounced activity than the monomer has, thus toxic activity may be related to the hydrolysis of phospholipids. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:53–58, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10060