Disulfide bond formation is the only known posttranslational modification o
f insect pheromone binding proteins (PBPs), In the PBPs from moths (Lepidop
tera), six cysteine residues are highly conserved at positions 19, 50, 54,
97, 108 and 117, but to date nothing is known about their respective linkag
e or redox status, We used a multiple approach of enzymatic digestion, chem
ical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, follo
wed by digestion with endoproteinase Lys-C to determine the disulfide conne
ctivity in the PBP from Bombyx mori (BmPBP), Identification of the reaction
products by on-line liquid chromatography-electrospray ionization mass spe
ctrometry (LC/ESI-MS) and protein sequencing supported the assignment of di
sulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The di
sulfide linkages were identical in the protein obtained by periplasmic expr
ession in Escherichia coli and in the native BmPBP. (C) 1999 Federation of
European Biochemical Societies.