福建产眼镜王蛇毒神经毒素的分离及活性测定

目的 从眼镜王蛇蛇毒中分离神经毒素，测定其活性和毒性。方法 应用CM—Sephadex C-50离子交换色谱，阶段梯度洗脱分离粗毒；用Sephadex G-50凝胶过滤纯化组分；用大鼠体外膈神经-膈肌标本鉴定分离组分对神经-肌肉接头的作用性质；观察神经毒素对乙酰胆碱(Ach)引起蛙体外腹直肌收缩的量效曲线的影响；以Meier法测定神经毒索对小鼠的半数致死量(LD50)。结果 眼镜王蛇粗毒经阳离子交换色谱后获得A～N共14个蛋白峰，经鉴定E、F、G，H、I、K等6个蛋白峰具有阻断神经一肌肉接头传导的作用，被鉴定为神经毒素，约占粗毒的48．3％；通过凝胶过滤对E、G和K组分进行初步纯化；E、G和K组分可使Ach引起的蛙腹直肌收缩的量效曲线平行右移，它们与N2-胆碱受体(N2-AchR)的解离常数(KD)分别为：145．87，73．53和20．04ng／mL；E、G和K组分小鼠静脉注射的LD50分别为：0．884，0．749和1．58mg／kg。结论 眼镜王蛇粗毒经离子交换色谱获得6个神经毒索组分，经鉴定E、G和K组分为N2-AchR的竞争性拮抗剂。

The Isolation and Charaterization of the Neurotoxic Fractions from Ophiophagus hannah Venom

Objective To isolate and charaterize the neurotoxic fractions from Ophiophagus hannah venom. Methods CM-Sephadex C-50 column was used to isolate the venom with step-wise elution. The fractions were purified by Sephadex G-50. Neurotoxicity was determined on isolated rat phrenic nerve-diaphragm preparations. Effects of neurotoxic fractions on acetylcholine-induced contractile response of the isolated frog rectus abdominis were observed. The LD-{50} of neurotoxic fractions was determined by Meier and Theakston method. Results Forteen fractions(A~N) were isolated from Ophiophagus hannah venom, six fractions(E, F, G, H, I and K) of which are neurotoxic and occupied the content of 48 3%. Fraction E, G and K produced a concentration-dependent parallel shift to the right of dose-response curve of acetylcholine applied exogenously with no change in the maximal asymptotic response. The dissolation constant K-D of N-2-AchR in fraction E, G and K were 145 87, 73 53 and 20 04 ng/mL, respectively. The LD-{50} of fraction E, G and K in mice(iv) were 0 884, 0 749 and 1 58 mg/kg respectively. Conclusion Six neurotoxic fractions were isolated from Ophiophagus hannah venom(from Fujian). Three fractions(E, G and K) were determined as the competitive antagonists of N-2-AchR.;