Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance.

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.