Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines β100 and β110 in porcine luteinizing hormone: Metastability and hypothetical isomerization of the two disulfide bridges of its β-subunit seatbelt

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Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, α and β, that are non-covalently associated. The heterodimer is stabilized by a region of the β-subunit called the “seatbelt” because it wraps around the α-subunit and it is fastened by a disulfide bridge between cysteines β26 and β110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines β26 and β110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines β100 and β110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysβ93 and β26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges β26–β110 and β93–β100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.