Purification, Characterization And Analgesic Activity Of The Neurotoxins From Ophiophagus Hannah Venom

The purification and characterization of the neurotoxins from OhVFourteen fractions(A~N) were isolated from Ophiophagus hannah Venom by CM-Sephadex C-50 column with step-wise elution. Among them six fractions(E, F, G, H, I and K) are determined neurotoxic on isolated rat phrenic nerve-diaphragm preparations and have the content of 48.3% in venom. They all produced a concentration-dependent parallel shift to the right of dose-response curve of acetylcholine applied exogenously on the isolated frog rectus abdominis with no change in the maximal asymptotic response like tubocurarine and were determined as the competitive antagonists of N2-AChR, so they are α-neurotoxins. The dissolation constant KD of fraction E, G and K with N2-AChR were 145.87, 73.53 and 20.04 ng/ml respectively. The LD50 of fraction E, G and K in mice (iv.) were determined as 0.884, 0.749 and 1.58 mg/kg respectively by Meier and Theakston method. Fraction K has higher potency on blocking the N2-AChR and lower toxicity.A neurotoxin OhV-K2 was purified from fraction K by CM-Sepharose Fast Flow column, Phenyl Sepharose High Performance column and Sephasil Peptide 5μm C18 ST 4.6/250 column on AKTA explorer system. OhV-K2 migrated as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) which showed that OhV-K2 was homogeneous. The molecular weight of OhV-K2 is determined by Image Master VDS system as 7776 dalton or so and fixed by ESI-MS as 7754 dalton. The 1~8 N-termination ammonic acid sequence of OhV-K2 is analysed by Edman degradation as TKCYITPD. 2. The analgesic activity of OhV-K2The hot-plate test showed that this neurotoxin increased latency time dose-dependently (from about 0.5 to 1.5 times) in mice when administered i.v. in the dose range of 0.025-0.4mg/kg. An i.v. dose of 0.025 mg/kg provided an analgesic effect comparable to that of 5 mg/kg morphine given by i.p.. Thus, OhV-K2 is about 4100 times more potent an analgesic agent than morphine on a molar basis. In the hot-plate test, the analgesic effect of OhV-K2 peaked at 5 hr and remained significant up to 12 hr after administration. OhV-K2 produces analgesia with a little development of tolerance and no addict. Similar analgesic action was observed in formalin test. But it had no antinociceptive effect on acetic acid-induced writhing in mice. The analgesic effect of OhV-K2(0.1 mg/kg) was blocked by atropine(1 mg/kg) and naloxone(6 mg/kg) in hot-plate and formalin test, but enhanced by nicotine.