平颏海蛇神经毒组分Ⅷ的分离纯化及毒理的初步研究

Isolation purification and toxicological identification of neurotoxin-fraction ⅩⅢ from the venom of Lapemis hardwickii

平颏海蛇毒腺提取物用CM-Sephadex C25柱层析,经醋酸铵缓冲液梯度洗脱,获得19个蛋白组分。经离体小鸡颈二腹肌标本检验,其中14个具有突触后神经毒作用。组分XⅡ再经Sephadex G 50和CM-Sephadex C 50层析纯化后得到组分XⅢ-2-4,该组分用Sephadex G 50过滤呈单一蛋白吸收峰,经三种不同类型的聚丙烯酰胺凝胶电泳鉴定为电泳纯蛋白。其等电点为8.3,表观分子量为8200。小鼠ipLD_(50)为94μg/kg(86～102μg/kg)。其对小鸡颈二腹肌标本的神经肌肉传导的阻断作用经反复冲洗360min,结果表明是难于逆转的。根据氨基酸组成分析、分子量测定以及对神经肌肉传导阻断的难逆转结果,推测平颏海蛇神经毒素XⅢ-2-4可能属于突触后长链神经毒。

The venom of Lapemis hardwickii has been separated by chrcmato-graphy on CM-Sephadex C 25 into 19 fractions.Among them 14 fractions were postsynaptic neurotoxic.Fraction ⅩⅢ,one of the neuro-toxic fraction,was purified by rechromato-graphy on Sephadex G 50 and CM-Sephadex C 50 and a purified toxin,fraction ⅩⅢ-2-4 was obtained.It was proved to be homogeneous by gel filtration on Sephadex G 50 and three different kinds of polyacrylamide gel eleclrophoresis Its molecular weight was about 8200 and isoelectric point was 8.3.The half lethal dose (ip)was 94μg/kg(86-102μg/kg).The blocking effect of fraction ⅩⅢ-2-4 on neuronuiscular transmission in isolated Chick biventer cervicis preparation was hardly reversible after repetitive washing within 6 hours.According to amino acid composition,molecular weight and the properties of neu-romuscular blockade,it is inferred that the fraction ⅩⅢ-2-4 may belong to a long chain postsynaptic neurotoxin.