Liang S, Xia Z, Xie J
Department of Biology, Hunan Normal University, 410081, Changsha, China.
Sci China C Life Sci. 1997 Oct;40(5):449-57. doi: 10.1007/BF03183581.
Huwentoxin-I, a neurotoxic peptide from the spiderSelenocosmia huwena, was synthesized by solid-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Boc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. The synthetic peptide was cleaved from the resin and deprotected by a 90 % TFA solution containing 5 % thioanisole, 3% ethanedithiol and 2 % anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The synthetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse phrenic nerve diaphragm preparation. The results indicated that the synthetic huwentoxin-I has the same chemical and conformational structure and biological activity as those of the native huwentoxin-I from the spider.
虎纹毒素-I是从蜘蛛虎纹捕鸟蛛中提取的一种神经毒性肽，采用芴甲氧羰基氨基酸五氟苯酯（Fmoc-AA-OPfp）通过固相法合成。羧基和羟基用叔丁基保护；赖氨酸和组氨酸的侧链用叔丁氧羰基保护；精氨酸的胍基用4-甲氧基-2,3,6-三甲基苯磺酰基保护，半胱氨酸的巯基用三苯甲基保护。固相载体为乙二胺-聚乙二醇-聚苯乙烯（DEA-PEG-PS）树脂。合成肽从树脂上切割下来，并用含有5%硫代苯甲醚、3%乙二硫醇和2%苯甲醚的90%三氟乙酸溶液脱保护。产物用二硫苏糖醇还原，然后与氧化型谷胱甘肽和还原型谷胱甘肽一起孵育以形成正确的二硫键连接。合成肽通过高效液相色谱法纯化，然后通过氨基酸组成和序列分析、肽图谱分析和核磁共振进行表征。使用分离的小鼠膈神经膈肌制备物通过电生理方法测试合成产物的生物活性。结果表明，合成的虎纹毒素-I与来自蜘蛛的天然虎纹毒素-I具有相同的化学和构象结构以及生物活性。
