新型桶形芋螺毒素BtIIIB的分离纯化、氨基酸序列测定及二硫键定位研究

采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtIIIB．采用氨基酸组成分析、质谱分析和Edman降解测定BtIIIB为15肽, 其氨基酸序列为: CCELPCHGCVPCCWP．结构中含有6个半胱氨酸, 形成3对分子内二硫键．采用部分还原的方法, 分步还原毒素中的二硫键, 用氰基化试剂衍生生成巯基, 然后在碱性条件下将衍生的产物进行裂解, 用MALDI-TOF MS测定裂解后片段的分子量, 确定了其二硫键的配对方式为Cys¹-Cys¹³, Cys²-Cys⁹, Cys⁶-Cys¹²的部分交叉式结构, 是芋螺毒素中比较特别的配对方式．

Purification, Sequence and Disulfide Bonding Pattern of a Novel Conotoxin BtIIIB

A peptide toxin, named conotoxin BtIIIB, has been isolated from the venom of Conus betulinus by gel filtration and reverse phase high performance liquid chromatography. The sequence of the peptide, CCELPCHGCVPCCWP, was determined by combination of mass spectrometry, amino acid analysis and Edman degradation. It contains 6 cysteines, forming 3 intramolecular disulfide bonds. In order to determine its disulfide bonding pattern, the toxin was reduced partially by TCEP tris-(2-carboxyethyl)phosphine] first. The formed —SH was cyanated with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) and the products were purified. Each pure cyanated product was cleaved with ammonia and analyzed with MALDI-TOF MS. Conotoxin BtIIIB is a pentadecapeptide with Cys¹-Cys¹³, Cys²-Cys⁹ and Cys⁶-Cys¹² three disulfide bonds.