The disulphide bond structure of thyroid-stimulating hormone β-subunit

Previously only one of the six disulphide bonds within the βsubunit of bovine thyrotropin (bTSHβ) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSHβ, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys))–Cys*& ; the second most reactive group of disulphide bonds involved the half-cystine residues Cys"', Cys"*, Cys'( and Cys"!& with the experimental results consistent with the assignment of disulphide bonds to Cys"'–Cys'( and Cys"*–Cys"!&. The least reactive group of halfcystine residues consisted of Cys#, Cys#(, Cys$", Cys&#, Cys)$ and