Abstract
A Phospholipase A2 termed as BSS PLA2 was purified from the venom of Bungarus sindanus sindanus (Sindhi Krait) through two steps: size exclusion chromatography on sephadex G-75 and reversed-phase chromatography on Vydac C18 HPLC column. The homogeneity and molecular mass of BSS PLA2 were analysed by SDS-PAGE and MALDI-TOF mass spectrometry. The N-terminal sequence of 32 amino acids of the BSS PLA2 revealed close resemblance with other species of bungarus snakes. The BSS PLA2 showed an optimum activity in the alkaline range (pH 8.0-9.5), while the optimum temperature stability was found to be 60 ° C. The effect of calcium ion (Ca2+) concentration revealed that in the absence of Ca2+ the enzyme showed some activity however, the enzymatic activity increases with increase in Ca2+ concentration. The addition of exogenous metal ions, including Mg2+ with Ca2+ increases, while that of Ba2+ with Ca2+ slightly decreases the enzymatic activity of BSS PLA2. Additionally, kappa bungarotoxin with the mass of 7358.5 Da was identified by MALDI TOF MS/MS from another peak of the same HPLC elution. Two of the tryptic peptides matched with kappa-6-bungarotoxin when searched through Mascot search engine.
Keywords: Bungarus sindanus sindanus, PLA2, Purification, kappa bungarotoxin, Mass spectrometry, Snake venom, Size exclusion chromatography, In-gel tryptic digestion, MALDI, Anti-venoms
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