Disulfide bonds of glycoprotein hormones. Their selective reduction in the beta subunits of bovine lutropin and thyrotropin.

Abstract

Treatment of the β subunit of either bovine lutropin (LH) or thyrotropin (TSH) at pH 8.2 by 5 mM dithioerythritol produces a set of partially reduced intermediates which, after alkylation with [14C] iodoacetate, can be separated by anion‐exchange chromatography. Fractions from LH‐β are obtained which represent molecules with 1, 2, 3 and 4 or 5 of the six disulfide bonds reduced. After full reduction, complete alkylation with non‐radioactive iodoacetate and subsequent isolation of tryptic peptides, the radioactivity of each carboxymethyl cysteine residue was determined. With LH‐β, the data show that disulfide bonds between half‐cystines 93–100 and 26–110 open sequentially and that a third bond is between half‐cystine 72 and either half‐cystine 23 or 88; a specific disulfide interchange appears to have occurred between the two last positions. As reduction proceeds, extensive interchange occurs which prevents further assignment of disulfides; the half‐cystines involved in disulfides 93–100 and 26–110, however, are not involved in the interchange. It is of interest that the assignments of bonds at 93–100 and 26–110 are in agreement with results of partial hydrolysis experiments from other laboratories, while the positions most involved in interchange are those where assignments from other laboratories differ. Under the same conditions of reduction, TSH‐β yielded fractions containing one bond open (positions 88–95, analogous to 93–100 in LH‐β) and small amounts of material in which reduction had occurred at positions 19–105 and 31–85 (analogous to 26–110 and 23–72 in LH‐β). The data indicate that during the initial steps of reduction, the β subunits of LH and TSH open in a similar fashion, though disulfide interchange may begin sooner in the case of TSH. The results strongly support the assignments of two of the six disulfides in β subunits and show that unequivocal assignment of the four remaining bonds remains to be achieved.