Purification, characterization and bactericidal activities of basic phospholipase A_2 from the venom of Agkistrodon halys (Chinese pallas)

摘要

Agkistrodon snake venoms contain a variety of phospholipases (PLA2), some of which are myotoxic. In this study, we used reverse-phase HPLC to purify PLA_2 from the venom of Agkistrodon halys. The enzyme named as AgkTx-II, a basic Asp49 PLA_2, has a molecular masses of 13,869.05. The amino acid sequence and molecular mass of AgkTx-II was identical to those of an Asp49 basic myotoxic PLA_2 previously isolated from this venom. Antibacterial activities were tested by susceptibility and broth-dilution assays. AgkTx-II exerted a potent antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. The MIC values of AgkTx-II ranged between 85 and 2.76 μM and was most effective against S. aureus, P. vulgaris, P. mirabilis (MIC of 21.25 μM) and B. pseudomallei (MIC of 10.25 μM). This AgkTx-II rapidly killed 5. aureus, P. vulgaris and B. pseudomallei in a dose-dependent manner. The effect of the AgkTx-II on bacterial membranes was evaluated by scanning and transmission electron microscopy. AgkTx-II caused morphological alterations apparent on their cellular surfaces, suggesting a killing mechanism based on membrane permeabilization and damage. Cytotoxicity was measured by XTT tetrazolium (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and lactate dehydrogenase (LDH) assays using U-937 cells (monocytes). The AgkTx-II did not affect cell viability up to 500 μM concentrations but cell death was evident at 1000 μM concentration after 24 and 48 h. Furthermore, the repeated exposure of AgkTx-II (2-14 μM) treated mice showed different tissue alterations, mainly at the brain and kidney; the toxicological potential of AgkTx-II remains to be elucidated. The AgkTx-II exhibits no hemolytic action even at high doses (10-100 μM) in human erythrocytes. However, the AgkTx-II is believed to exert its bactericidal effect by permeabilizing the bacterial membrane by forming pores. In addition, the basic PLA_2 AgkTx-II displays a bactericidal effect, which may be either dependent or independent of catalysis.