Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I ) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by m atrix-assisted laser desorption ionization time-of-flight mass spectroscopi c analysis. To map the disulfide bonds, native SHL-I was proteolytically di gested. The resulting peptides were separated by reverse phase high-perform ance liquid chromatography. Matrix-assisted laser desorption ionization tim e-of-flight mass spectroscopic analysis indicated the presence of one disul fide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2- carb oxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performan ce liquid chromatography. Four M Guanidine-HCl was found to increase the yi elds of partially reduced peptides prominently. The free thiols were carbox amidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of t he partially reduced and alkylated SHL-I with that of the intact peptide. F inally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys1 4, Cys7-Cys19, Cys13-Cys26. (C) 1999 Elsevier Science Inc. All rights reser ved.