Isolation and characterization of phospholipase A1 from Vespa tropica ducalis / 中草药

ABSTRACT

Objective: To find an effective method to isolated phospholipase from Vespa tropica ducalis and characterize its biological activities to support the pathogenic mechanism research and officinal value exploitation in the future. Methods: The component with phospholipase activity was isolated by successive gel filtration (Sephadex G-75, supferfine) and heparin affinity chromatography steps (Hitrap Heparin HP). The protein was identified by peptide mass fingerprinting, N-terminal amino acid determination and blast analysis, as well as phospholipase A1 (PLA1) activity monitor. Plasma recalification time test was employed to detect the effect of Vtp32 on coagulation. Results: A protein with phospholipase activity was orderly separated and purified from V. tropica ducalis venom using gel filtration and heparin affinity chromatography. The purified protein was homogenous on the SDS-PAGE gel with relative molecular mass of 32 000, so it was termed as Vtp32. Peptide mass fingerprinting assay and N-terminal amino acid sequence blast result revealed that Vtp32 showed high homologous with PLA1 from wasp of Vespa genus. In addition, Vtp32 hydrolyzed the sn-1 ester linkage of phospholipids. These results indicated that Vtp32 was PLA1 from V. tropica ducalis. Vtp32 hydrolyzed phosphatidylcholine, and the hydrolysis product can lyse human erythrocytes. Vtp32 delayed the recalification time of human plasma and hence had anti-coagulation activity. Conclusion: PLA1 is widely existed in the venom from V. tropica ducalis. Gel filtration followed by heparin affinity chromatography is an effective isolation strategy for the purification of PLA1. The results show that V. tropica ducalis PLA1 has hemolytic and anticoagulative activity.