Purification and characterization of phospholipase A from Bungarus multicinctus venom.

Abstract

Phospholipase A was purified 16.3-fold in terms of specific activity from the venom of Bungarus multicinctus by ion exchange chromatography on a CM-Sephadex C-25 column followed by gel filtration on a Sephadex G-75 column. The overall enzyme yield was 3.5% from the crude venom. The molecular weight and isoelectric point of the purified enzyme were estimated to be 14,000 and 7.9 by electrophoresis on SDS-polyacrylamide gel and on polyacrylamide gel for isoelectric focusing, respectively. The enzyme was a single polypeptide chain consisting of 118 amino acids. Its N- and C-terminal residues were asparagine and glutamine, respectively. The enzyme was stable in the pH range of 2--10 and retained full activity after incubation for 24 h at 37 degrees C. The optimum hydrolysis of egg yolk phosphatidyl choline was observed at pH 8.5 and the specific activity was 1,450 units per mg of the enzyme. The enzyme was inactivated by treatment with p-bromophenacyl bromide, accompanied by the loss of one of two histidine residues.