Assignment of the three disulfide bridges of huwentoxin-I, a neurotoxin from the spider selenocosmia huwena.

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spider Selenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact and in situ S-carboxymethylated toxin. In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys2-Cys17, Cys9-Cys22, and Cys16-Cys29.