摘要
多肽毒素存在于自然界的很多生物物种中，生物学活性是多肽毒素研究一个极为重要的方面。综述了多肽毒素的生物学活性及相应的研究方法。
通过离子交换和反相-HPLC从海南捕鸟蛛粗毒分离了海南捕鸟蛛毒素-Ⅳ，MALDI-TOF测定其分子量为3988.70，通过氨基酸组成分析、Cys烷基化修饰、Lys-C酶水解后HPLC分离片段序列测定和羧肽酶-Y C-端测序确定了其氨基酸序列为：ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI，该序列与从虎纹捕鸟蛛分离的HWTX-Ⅳ只相差7个氨基酸残基。其中6个Cys形成3对二硫键，在pH 3的条件下利用TCEP对海南捕鸟蛛毒素-Ⅳ进行了部分还原，收集RP-HPLC的洗脱峰，用碘乙酰胺烷基化修饰游离的-SH，经质谱鉴定后进行氨基酸序列测定，由此确定了Cys2-Cys17、Cys9-Cys24和Cys16-Cys31形成了1-4、2-5和3-6的二硫键配对方式。小鼠急性毒性实验确定海南捕鸟蛛毒素-Ⅳ的LD_(50)为0.20±0.07 mg／kg，而对美洲蜚蠊却没有影响。海南捕鸟蛛毒素-Ⅳ能使电刺激引起的大鼠输精管收缩产生先兴奋后抑制的作用，而对电刺激诱导的小鼠膈神经-膈肌收缩却只产生抑制作用，浓度为1.0×10~(-6) mol／L时的阻断时间为18.1±0.2 min(n=5)，同时它对由去甲肾上腺素引起的输精管收缩和直接电刺激肌肉引起的膈肌收缩没有影响，推测其具有神经专一性。电生理实验表明海南捕鸟蛛毒素-Ⅳ能完全阻断TTX-敏感型的钠通道，而对钠通道的激活和失活没有影响，从而推测其是一种位点-1毒素。由以上结果可推断海南捕鸟蛛毒素-Ⅳ为一种新型的作用于神经细胞TTX-敏感型钠通道位点-1的阻断剂。通过固相化学合成法合成了海南捕鸟蛛毒素-Ⅳ，并对其氧化复性条件进行了摸索，确定了0.1 mol／L Tris-HCl和0.1 mol／L NaCl缓冲液、pH 8.0、样品浓度为0.1g／L、
中文摘要
5二。1/L GSH、0.5 fnmol/L GSSG为最适条件。复性样品经质谱、与天
然毒素混合进样IIPLC分析和生物学活性分析，确定复性的海南捕鸟蛛
毒素一Iv与天然毒素在结构和功能具有一致性。通过2D一NMR技术已初步
确定了海南捕鸟蛛毒素一IV的空间结构由两个p一折叠和4个p一转角组
成，三对二硫键稳定这一构象。通过与HWTx一IV和比conot。xin GlllA
的比较分析，确定了转角一Iv为海南捕鸟蛛毒素一Iv的活性相关区域，
而Ly 5 27为活性关键残基。
从虎纹捕鸟蛛粗毒分离纯化到了虎纹捕鸟蛛毒素刁工I(H wTx一IH)的
天然突变体(m一IIWTX一工H)和凝集素刁(SHL一I)的天然突变体(SLH一H).
两种突变体的氨基酸序列都只比相应的毒素多肤少了C一端的Trp残基，
而其它序列完全相同。两者的序列分别为:DcAGYMREcKEKLccsGYvcss-
RWKWCVLPAP和GCLGDKCDYNNGCCSGYVCSRTWKWCVLAGP。通过比较研究它们
的生物学活性，发现虎纹捕鸟蛛毒素一IH的天然突变体没有虎纹捕鸟蛛
毒素一IH的使输精管收缩加强的作用，从而推测其c一端残基为生物学
活性相关残墓。凝集活性实验表明凝集素班及其天然突变体具有相同的
凝集活性，同时ID一NMR分析确定两者空间结构相似，从而推测其C一端
对于凝集素一I不是活性相关残基。
A neurotoxic peptide (HNTX-IV) was purified from the venom of the spider Selenocosmia hainana with combination of ion-exchange and reverse-phase HPLC. The complete amino acid sequence (35 residues) was obtained by using automated Edman degradation, which is agreement with the molecular mass 3 988.70Da by mass spectrometry and the amino acid analysis. The amino acid sequence of HNTX-IV is similar to that of HWTX-IV except only seven residues. HNTX-IV was partially reduced by TCEP at pH 3, and the intermediates were isolated by revers-phase HPLC. Alkylation of free thiols, followed by sequencer analysis, enabled all three bridges to be identified (Cys2-Cys 17, Cys9-Cys24 and Cysl6-Cys31). HNTX-IV was synthesized by using Fmoc-chemistry methodology and the renatured peptide had the same biology activity as the native toxin. HNTX-IV indues lethal activity on rats but has no effect on cockroches. The LD50 of rats is (0.20 ± 0.07) mg/kg. HNTX-IV courses enhancement of the twitch response induced by electronical stimulation of vas deferens and then inhibition completely. HNTX-IV only inhibits the constraction of phrenic nerve hemidiaphragm preparations. HNTX-IV has no effect on the rhythmic contraction of vas deferens triggered by adding noradrenaline and the twitch response of hemidiaphragm induced by directly stimulating muscale, which indicats that is a nerve specific toxin. Electrophysiological studies showed that HNTX-IV inhibits TTX-S sodium current completely in NG108-15 cells
and has no effect on the activation and inactivation of sodium current, which suggests HNTX-lV is a site-1 toxin affecting the sodium channel through a mechanism quite similar to that of TTX. The three-dimensional structure of HNTX-IV has been determined by 2DTSJMR. The resulting structure is composed of a double-stranded antiparallel (3-sheet (L22-S25 and W30-Y33) and three turns (E4-K7, PI 1-D14, K18-K21 and R26-R29) and belongs to the inhibitor cystine knot structural family. Comparison with u-conotoxin GIIIA and HWTX-IV shows that the positively charged residues of loop IV (residue 26-29), especially residue Lys 27, must play a crucial role in its binding to
neuronal tetrodotoxin-sensitive voltage-gated sodium channel.
Two peptide components, named m-HWTX-III and SHL-II, were isolated from the crude venom of the spider Selenocosmia huwena. The sequences of the two peptides are similar to these of HWTX-III and SHL-I besides lack of the C-terminal tryptophan residue repectively. m-HWTX-III neither causes paralysis in cockroaches (p,americana) nor enhances electrically induced contraction of rat vas deferens, while as HWTX-III does. The result indicates that Trp33 is a key residue related to the biological function of HWTX-III. SHL-II and SHL-I, However, have the same haemagglutination activity to each other, which suggests that the C-terminal of SHL-I is not important to its haemagglutination activity.
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