Purification and properties of two phospholipase A2 enzymes from berg adder (Bitis atropos) venom
Two phospholipases A2 (PLA2-1 and PLA2-2) of berg adder (Bitis atropos) venom were purified by successive chromatography on Cellex-CM, Sephadex G-75 and Bio-Gel P-100 columns. PLA2-1 is an acidic PLA2 (pl 6.6) with a molecular weight of 29 393.12 as determined by electrospray ionization mass-spectrometry (ESI-MS). When treated with a reducing agent, it split into two chains of about 15.7 kDa and 13 kDa, as revealed by sodium dodecyl sulphate-polycrylamide gel electrophoresis. Although PLA2-1 displayed cytotoxic activity in vitro on Be-11 melanoma cells (EC50=76.6 μg ml-1), no toxic effects were noted in mice. The molecular weight of PLA2-2 as determined with ESI-MS is 14 034.89. This enzyme, a basic monomeric PLA2 (pl 9.3), induced neurotoxic symptoms in mice. An intra-peritoneal LD50 of 1.7 mg kg-1 was calculated. Its N-terminal 19 amino acid sequence was Asn-Leu-Tyr-Gln-Phe-Gly-Lys-Met-Ile-Ser-His-Lys-Thr-Asn-Asn-Gly-Pro-Leu-Ala. This is the first report of the isolation of a dimeric as well as monomeric PLA2 enzyme from the venom of Bitis atropos.