Open AccessAssignment of the disulfide bonds of huwentoxin‐II by Edman degradation sequencing and stepwise thiol modification
Author(s) -
Shu Qin,
Huang Renhuai,
Liang Songping
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2001.02109.x
Subject(s) - edman degradation , chemistry , proteolysis , peptide , thiol , cysteine , disulfide bond , thioamide , stereochemistry , combinatorial chemistry , peptide sequence , biochemistry , enzyme , gene
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 (ΔSer), 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.