Neurotoxic Activity Of Phospholipase A₂ From Agkistrodon Blomhoffii Ussurensis Snake Venom

The conditions of purification of Gln49-PLA₂ from Agkistrodon blomhoffii ussurensis snake venom were optimized. The pure Gln49-PLA₂ could be obtained by Hitrap SP cation exchange and Superdex 75 gel-filtration chromatography. The highly purified 0.6 mg/ml Gln49-PLA2 with 5% (w/v) fucose added, stored at -20℃, was retained its anticoagulant effect about 80.6% of the fresh one after 14 days.The Gln49-PLA2 showed weak lethal toxic (LD50 is 18.2 mg/kg). Hot-plate tests showed that it obviously induced an increase of the pain threshold upon intoxicated 615 mice compared with the control group and its analgesic activity was dose-dependent, suggesting it is a neurotoxin. Naloxone could antagonize the analgesic effect, which was implying the mechanism of action of the Gln49-PLA2 is correlative with opioid acceptors. The electrophysiology studies revealed decreases of the action potential and the nerve conduction velocity on isolated hoptoads' sciatic nerve, indicating the Gln49-PLA2 most probably effected on ion channels.Patch-clamp experiments showed the Gln49-PLA2 could reduce the currents and conductance of potassium channels of SHSY5Y cell, but has no effect on sodium channels. The acticity of Gln49-PLA2 could not be effected by CsCl, suggesting that it did not act on the delayed rectifier potassium channel. Naloxone could antagonize the effect of Gln49-PLA2, indicating that the Gln49-PLA2 acted on the opioid receptors directly or indirectly through the endogenous opioid peptides. However, opioid receptors could activite different kinds of potassium channels which were restrained by the Gln49-PLA2, implying the Gln49-PLA2 effected on opioid receptors indirectly.