Effect of Radioiodination on the Structural Integrity, Receptor and Antibody-Binding Activity and Circulatory Behavior of Human Choriogonadotropin

Abstract

Human choriogonadotropin (hCG) was labelled with ¹²⁵I using a low temperature (4°C), minimal chloramine-T (10 µg) concentration and 20-sec oxidization. The structural integrity of labelled hCG was analyzed immediately after the labelling as well as after storage for 7 and 14 days at 4 or –15°C by gel filtration on a Sepharose 6B and Sephadex G-200 column and by polyacrylamide gel electrophoresis. Gel filtrations of freshly labelled hCG revealed a single radioactive peak (K_av = 0.52 and 0.33) corresponding to the intact unlabelled hCG. Polyacrylamide gel electrophoresis also showed a high homogenity of labelled hormone. The capability of freshly labelled hCG to bind to LH(hCG) receptor was 51% and to anti-hCG γ-globulin Sepharose 4B, 94%. Storage for 14 days at 4°C caused a complete change of the molecule to a smaller conformation (K_av = 0.62 and 0.41) accompanied by a marked reduction in the binding to LH(hCG) receptor (12%). The binding to specific antibody remained high (75%). No change in the structure was found when the labelled hormone was stored for 14 days at – 15°C. The hormone also exhibited high binding to the LH(hCG) receptor (45%) and to specific antibody (86%). SDS-polyacrylamide gel electrophoresis indicated that over 90% of the radioiodine present in labelled hCG was associated with the α-subunit. No difference was found in the disappearance rate of freshly labelled and fresh unlabelled hCG from the circulation of the rat after a single intravenous injection. The rate of disappearance of the structurally changed hormone was, however, markedly increased and was found to be due to the higher uptake of the hormone by the kidneys and liver. The present observations indicate that hCG retains well its structural integrity and capability to bind to LH(hCG) receptor and specific antibody when radioiodinated with the chloramine-T method. However, there occurs a rapid structural change in labelled hormone molecules under storage at 4°C leading to a smaller conformation, a marked reduction in its binding capability to LH(hCG) receptor and altered metabolic clearance in the rat.