33] Cobra venom phospholipase A2: Naja naja naja

Abstract

Phospholipase A2 (PLA2) is a major component of most snake venoms and can be purified in large quantities from this source. Purification of the enzyme is complicated by the presence of multiple isozymes in many species. The venom from the Indian cobra (Naja naja naja) contains as many as 14 PLA2 isozymes. This chapter describes the purification of an acidic phospholipase A2 from this source. The standard assay mixture contains 5 mM egg phosphatidylcholine, 20 mM Triton X-100, and 10 mM CaCl2. The appropriate volume of egg phosphatidylcholine solution is measured into a homogenization tube. The sample is dried first under a stream of nitrogen then under vacuum until all the chloroform has evaporated. The appropriate amounts of Triton X-100 and CaCl2 are added, and the solution is brought to the final volume by addition of deionized water. The assay is also routinely performed with commercially available dipalmitoylphosphatidylcholine as a substrate. The purification scheme is carried out at room temperature. Protein concentrations are determined by using the correction factor for Naja naja naja phospholipase A2. The Lowry assay overestimates the protein concentration for this enzyme when bovine serum albumin is used as a standard. Therefore, protein values obtained by this method are multiplied by a factor of 0.66 to obtain the correct value. This procedure yields a PLA2 preparation that displays a single protein band on analytical isoelectric focusing, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and Ouchterlony double-diffusion precipitation tests.