An Immunological Approach to the Study of Protein Conformational Heterogeneity: Its Application to Growth Hormone*

Abstract

The study of multiple forms of a hormone due to changes in its secondary or tertiary structure (conformational heterogeneity) has been restricted to methods of physical chemistry which require relatively large quantities of purified preparations and has precluded such studies on circulating hormones. The present resport describes an immunological approach to the study of GH [growth hormone] conformational heterogeneity based on the simultaneous use of 2 homologous RIA [radioimmunoassay]: one which recognizes porcine (p) GH in its native conformation (pGH RIA) and a 2nd (pB RIA) for a 55-residue S-aminoethylated CNBr fragment (pB) of pGH (corresponding to residues 126-180 of human GH). In the pGH RIA, pB was nonreactive, whereas pGH displacement of [125I]iodo-pB from anti-pB serum required about 103 M excess and was nonparallel to pB. Treatment of pGH with urea plus mercaptoethanol (ME), which would be expected to affect the conformation of the GH molecule, increased its immunoreactivity 68-fold in the pB RIA and its dilution curve became parallel to that of pB. Similar treatment of porcine pituitary extract increased its immunoreactivity 97-fold in pB RIA with a corresponding slope change. In contrast, urea plus ME decreased pGH and pituitary extract immunoreactivity by 84 and 92%, respectively, in the pGH RIA. The pB RIA/pGH RIA ratio of immunoreactivity for pGH and for pituitary extract increased 795-fold and 569-fold, respectively, after combined treatment. Lesser increases were observed after urea or ME treatment alone. In conditions where alterations in GH conformation were suspected (highly purified, aged human GH preparations which were no longer satisfactory as radioiodinated tracers for RIA), calculation of the pB RIA.hGH RIA ratio of immunoreactivity indicated increased values. Based on these results, a model is proposed in which unfolding of native hormone, with concomitant exposure of the antigenic determinant of pB, hidden within pGH, leads to increased immunoreactivity in the pB RIA and decreased immunoreactivity in the pGH RIA. In this model, the pB RIA/pGH RIA ratio of immunoreactivity would serve as an index of the state of hormonal unfolding.