Conformational stability and in vitro bioactivity of porcine luteinizing hormone

Abstract

Temperature-dependent dissociation of porcine luteinizing hormone (pLH) and of two of its glycoforms was studied by a combination of SDS-PAGE and micro-scale size-exclusion HPLC in parallel with the study of co-operative folding by high-sensitivity differential scanning calorimetry (HS-DSC). The transition temperature of dissociation of pLH at pH 7.0 as quantified by SDS-PAGE, HPLC and residual activity in radioreceptor assay was found to match exactly the transition temperature of its unfolding as measured by HS-DSC. Free α- and β-subunits did not exhibit any unfolding transition in the same conditions. The microcalorimetric data for two pLH isoforms exhibiting different glycosylations were identical to those of a preparation of non-separated isoforms. It is concluded that: (a) free subunits exhibit no co-operative folding (i.e. no stable three-dimensional structure) and co-operative folding occurs only in αβ heterodimers; (b) the co-operative folding is responsible for the stability of the association of subunits; and (c) the heterogeneity of carbohydrate chains does not affect the stability of folding and association of subunits. The fastening of the 'seat-belt' of the β-subunit embracing the α-subunit by the Cysβ26-β110 disulfide bridge had been postulated to play a role in the preservation of the dimeric structure of gonadotropins. The present work shows that dissociation of subunits is directly related to their loss of common co-operative folding. Copyright © 2001 Elsevier Science Ireland Ltd.