眼镜王蛇毒组分Ⅶ-2的纯化及其毒性鉴定

眼镜王蛇毒用CM-Sephadex C-25柱层析、醋酸铵缓冲液洗脱,获得20个蛋白组分。Ⅷ峰经CM-C 32重层析后获得组分Ⅷ-1与Ⅷ-2,组分Ⅷ-2经Sephadex G-50滤过后呈单一吸收峰。用三种不同类型的聚丙烯酰胺凝胶电泳鉴定组分Ⅷ-2为电泳纯蛋白。分子量为7,827;等电点约为pH 7.6。小鼠ip LD₅₀为0.155 mg/kg(0.146～0.165 mg/kg)。用离体小鸡颈二腹肌标本表明组分Ⅷ-2是属于突触后神经毒,其阻断作用在反复冲冼6小时内是相当难逆转的。从氨基酸组成测定结果的分析、分子量、双向免疫扩散、LD₅₀的比较以及阻断神经肌肉传递的难逆转性,可推测眼镜王蛇毒组分Ⅷ-2是与α-银环蛇毒素相似的难逆转性长链突触后神经毒素。

PURIFICATION AND TOXICOLOGICAL IDENTIFICATION OF FRACTION Ⅷ-2 FROM THE VENOM OF OPHIOPHAGUS HANNAH

The Ophiophagus hannah venom has been fractionated by chromatography on CM-Sephadex C-25 into twenty fractious. Fraction Ⅷ-2 was purified by gel filtration on Sephadex G-50, ion-exchange rechromatography on CM-Cellulose 32 and reuse of Sephadex G-50. Homogeneity was proved by three different kinds of polyacrylamide gel electrophoresis. Its molecular weight, calculated from the measurement in SDS-polyacrylamide gel electrophoresis, is 7,827, The isoelectric point is about 7.6 by isoelectric focusing. The LD₅₀ for mice (i p) is 0.155 mg/kg (0.146-0.165 mg/kg).Fraction Ⅷ-2 was shown to block the neuromuscular transmission of chick biventer cervicis muscle and inhibit the acetylcholine response of the muscle after neuromuscular blockade. No effect on the response of the muscle to direct stimulation or to KCI was observed. This indicates that fraction Ⅷ-2 is a postsynaptic neurotoxin, Its blocking action was hardly reversible after repetitive washing in 6 hours.From comparison between fraction Ⅷ-2 and α-Bungarotoxin in the amino acid composition analysis, molecular weight determination, double immunodiffusion, LD₅₀ and the properties of neuromuscular blockade, it is inferred that fraction Ⅷ-2 is a long postsynaptic neurotoxin which is similar to α-Bungarotoxin.