Purification,characterization And Analgesic Activity Of The Neurotoxin From Naja Naja Atra Venom

Objective: To isolate and purify the neurotoxic fraction from Naja naja atra venom （from Jiangxi），and to study its characterization of receptor kinetics and analgesic activity.Methods:1.The two-step rapid separation method including SP Sepharose Hign Performance cation-exchange chromatography and Superdex30gel filtration chromatography was adopted to isolate and purify the neurotoxic fraction from Naja naja atra venom（from Jiangxi）. The fraction was determined as neurotoxin on the basis of the experiment of isolated rat phrenic nerve-diaphragm preparations. It was identified as the purity by SDS-polyacrylamide gel electrophoresis (Tris-Tricine system).2.The LD50of neurotoxic fraction was determined by intraperitoneal injection in mice with the method of Meier．The drug-receptor interactions of cobra neurotoxin with frog rectus abdominis N2-receptor were studied and compared with that of vecuronium bromide (Vec).3.The analgesic action of cobra neurotoxin was observed by the methods of tail electric stimulation-vocalization test, hot-plate test and acid-writhing test in mice.4.The7-day consecutive dosing method was chosen to observe the tolerance of cobra neurotoxin. The naloxone precipitated withdrawal test was used to detect the dependence of cobra neurotoxin.Results:1.The venom of Naja naja atra（from Jiangxi）was fractioned on SP Sepharose Hign Performance cation-exchange chromatography into twenty-one different protein peaks. The protein peak IX named NNAV-IX was further purified by gel filtration chromatography on Superdex30. The protein peak IV named NNAV-IX4was identified as the electrophoretically pure neurotoxin whose molecular weight was about15kDa.2.The approximate LD50of NNAV-IX4by intraperitoneal injection was0.501mg/kg in mice with method of Meier. The cumulative dose-effect curves were parallel shift to the right under the action of NNAV-IX4. The pA2values of NNAV-IX4and Vec were7.33and6.48respectively. The KD values of NNAV-IX4and Vec were0.0512μ mol/L and0.330μmol/L respectively.3.The analgesic actions of NNAV-IX4were dose-dependent and time-dependent，which effected at2h and peaked at5h in mice. It was showed that NNAV-IX4could obviously increase the pain thresholds in mice compared with NS group for all three kinds of pain models (P<0.05). Especially in the tail electric stimula-tion-vocalization test, the pain thresholds of NNAV-IX4group increased by526.43%, which were tested5h after administration. By contrast, the pain thresholds of Dolantin group increased by171.63%only, which were tested1h after administration.4.The pain thresholds of NNAV-IX4group, which were tested5h after administra-tion，increased continuously and peaked on7th day compared with daily baseline pain thresholds(P<0.05). The pain thresholds of Morphine group, which were tested1h after administration, increased only on the first and second day. However, there was no difference between before and after administration in the following five days. The loss of weight and jumping times of Morphine group after injecting naloxone were different significantly compared with NS group(P<0.05), but there was no remarkable difference between NNAV-IX4group and NS group.Conclusions:1.NNAV-IX4separated from Naja naja atra venom (from Jiangxi) with two-step method is a electrophoresis pure neurotoxin.2.The characterization of NNAV-IX4is a competitive antagonism for N2-AChR, and its antagonistic ability is greater than vecuronium bromide.3.The analgesic action that NNAV-IX4produce is remarkable, which take effect and reach peak-time slowly.4.NNAV-IX4is not easy to result in tolerance and addiction.