The disulphide bond structure of 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 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 Cys85. The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSH beta 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-Cys85. 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 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.
Fairlie
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WD
Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Stanton
P G
PG
Hearn
M T
MT
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D013972:Thyrotropin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D013972:Thyrotropin; D002918:Chymotrypsin; D014357:Trypsin