This project focuses on the role of carbohydrate structures of human chorionic gonadotropin (hCG) in the subunit association kinetics, purification of pregnant mare serum gonadotropin, development of sub-nanomole sugar analysis of glycoproteins and of a highly specific and sensitive two sites immunoradiometirc assay of hCG. A. The carbohydrate moieties in hCG are not essential for the association of subunits as studied by the enhancement of 1-anilino-naphthyl-8-sulfonate (ANS). They do influence the rate of association. The Scatchard analysis of hormone: ANS binding reveals that hCG and deglycosylated hCG (HF-hCG) give the following apparent dissociation constant (KD= muM) and number of binding site (n): KD=3.7, nl=1 for hCG; KD= 2 and 8, nl=0.5 and 0.3 for HF-hCG. Kinetic data indicates that the subunit association begins with a second-order rate (400 m-1min-1 for hCG and 5700 M-l min. -1 for HF-hCG) as the rate-limiting step followed by the slow first order conformational transition of the alpha-beta complex. The influence of carbohydrate appears to reside at the first step. B. Three conventional column chromatography steps have been employed to obtain four potent fractions active in ovarian receptor binding. SDS-polyacryl amide gel electrophoresis has shown two bands in all four fractions which migrated at the positions of 25K and 48K daltons as compared to hCG at 24K and 35k. A sensitive and reliable method of monosaccharide analysis has been developed. The method is based on a reverse-phase high performance liquid chromatography to separate perbenzoylated methylglycosylic derivatives of sialic acids, N-acetyl-glucosamine, N-acetyl- galactosamine, galactose, mannose and fucose which are prepared from acid methanolysis of glycoproteins followed by benzoic anhydride treatment. C. A polyclonal antibody based on two-site immunoradiometric assay for hCG has been developed. The method is highly specific and sensitive for hCG assay in biological fluids, and is devoid of false positive derived from protease activities. Our system is capable of monitoring a wide variety of hCG molecules and potentially important for laboratory and clinical investigation.
