Conformational Studies of Ovine Lutropin (Luteinizing Hormone) and Its Native and Chemically Modified Subunits by Circular Dichroism and Ultraviolet Absorption Spectroscopy

Conformational features of ovine lutropin and its subunits were investigated as well as cyanogen-bromide-treated subunits and reconstitted dimers. Spectrophotometric titrations of tyrosine residues indicated that all the tyrosines could be ionized in lutropin and the subunits, but a least three of the tyrosines ionized abnomally in native hormone, two in the isolated α and none in the isolated β subunits. When unfolded at pH 2, the hormone exhibited a reversible difference spectrum corresponding to the exposure of approximately three buried tyrosine residues as estimated by thermal perturbation. Amplitudes of the difference spectra were correlated to the specific biological activity of the starting materials. The total number of tyrosine residues per mole of protein in agreement with sequence data was determined by magnetic circular dichroism. These spectra were shifted toward the shortest wavelengths, as for the ultraviolet absorption spectra, when the pH was lowered from 7 to 2. Near-ultraviolet circular dichroism was correlated to those results and both tyrosine residues and disulfide bridges were found to contribute to the negative 280-nm circular dichroic band. The foldings of lutropin and its isolated subunits were estimated in terms of only three structures, α-helix, β-sheet and aperiodic (irregular) structure from least-squares fitting of computed far-ultraviolet circular dichroism to experimental circular dichroism. The best fit for native hormone was 8%α-helix, 35%β-structure and 57% aperiodic. This fitting was in good agreement with the predicted conformation from amino acid sequence whith yielded 6%α-helix, 41%β structure and 52% aperiodic. During the acid unfolding, Which led to a biologically inactive hormone, most of the α-helix structure was lost and the β structure content was significantly decreased with a corresponding increase of aperiodic structure, indicative of a limited unfolding. Both subunits were found to participate to the unfolding. As compared with the predicted folding of the subunits in the native hormone, the isolated α subunit had essentially a lower β sheet content and the isolated β subunit a lower helical content. Refolded lutropin, obtained by raising the pH from 2.8 to 5.3, or recombined dimers from intact or cyanogen-bromide-treated subunits, which were biologically active, showed essentially the same conformational features as the native hormone. From the conformational criteria, cyanogen-bromide-treated subunits were indistinguishable from the intact subunits.