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.

Key words: conotoxin, Conus betulinus, disulfide bonding pattern