Shu Q, Huang R, Liang S
Life Sciences College, Peking University, Beijing, China.
Eur J Biochem. 2001 Apr;268(8):2301-7. doi: 10.1046/j.1432-1327.2001.02109.x.
A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.
开发了一种将埃德曼降解法与硫醇修饰相结合的新策略，用于确定虎纹毒素-II（HWTX-II）的三个二硫键。HWTX-II是从蜘蛛虎纹捕鸟蛛毒液中纯化得到的一种杀虫肽。在天然HWTX-II的埃德曼降解过程中，半胱氨酸的苯硫代乙内酰脲（Pth）衍生物和消除产物脱氢丙氨酸（ΔSer）可在半胱氨酸循环中观察到。这两种产物的出现表明HWTX-II的二硫键已断裂，并且产生了后半胱氨酸的游离硫醇基团。如果新生硫醇通过4-乙烯基吡啶逐步修饰，那么从测序信号中可以获得有关HWTX-II二硫键性质的信息。使用这种方法，HWTX-II的二硫键被确定为Cys4-Cys18、Cys8-Cys29和Cys23-Cys34，这与来自同一蜘蛛的神经毒性肽HWTX-I中的二硫键不同。使用这种策略，可以通过测序和n-1次修饰来确定小蛋白质的二硫键，其中n是蛋白质中二硫键的数量。HWTX-II二硫键的上述确定通过HWTX-II胰蛋白酶片段的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）得到了证实。通过MALDI-TOF MS监测HWTX-II的蛋白水解动力学，观察到了蛋白水解过程中的一些二硫键互换。
