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

Previously only one of the six disulphide bonds within the β-subunit 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 Cys⁸⁸-Cys⁹⁵; the second most reactive group of disulphide bonds involved the half-cystine residues Cys¹⁶, Cys¹⁹, Cys⁶⁷ and Cys¹⁰⁵ with the experimental results consistent with the assignment of disulphide bonds to Cys¹⁶-Cys⁶⁷ and Cys¹⁹-Cys¹⁰⁵. The least reactive group of half-cystine residues consisted of Cys², Cys²⁷, Cys³¹, Cys⁵², Cys⁸³ and Cys⁸³. The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSHβ derivative in which only the half-cystine residues Cys³¹, Cys⁸⁵, Cys⁸⁸ and Cys⁹⁵ were labelled enabled the assignment of a previously uncharacterized disulphide bond to Cys³¹-Cys⁸³. The remaining two assignments, Cys²-Cys⁵² and Cys²⁷-Cys⁸³, 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 TSHβ, owing to the combination of the high resolution of the reversed-phase HPLC peptide mapping procedures and the sensitivity of the fluorimetric detection method.