The disulphide bond structure or thyroid-stimulating hormone beta-subunit

Previously only one of the six disulphide bonds within the beta-subunit of bovine thyrotropin (bTSH beta) 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 bTSH beta, 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(88)-Cys(95); the second most reactive group of disulphide bonds involved the half-cystine residues Cys(16), Cys(19), Cys(67) and Cys(105) With the experimental results consistent with the assignment of disulphide bonds to Cys(16)-Cys(76) and Cys(19)-Cys(105). The least reactive group of half-cystine residues consisted of Cys(2), Cys(27), Cys(31), Cys(52), Cys(83) and Cys(85). The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSH beta derivative in which only the half-cystine residues Cys(31), Cys(85), Cys(88) and Cys(95) were labelled enabled the assignment of a previously uncharacterized disulphide bond to Cys(31)-Cys(85). The remaining two assignments, Cys(2)-Cys(52) and Cys(27)-Cys(83), were made by comparison with the recently published human chorionic gonadotropin crystal structure. The flexibility of the double-labelling approach used in these studies demonstrates that only very small quantities are required for proteins containing an extensive number of half-cystine residues such as TSH beta, owing to the combination of the high resolution of the reversed-phase HPLC peptide mapping procedures and the sensitivity of the fluorimetric detection method.