Subunits of human chorionic gonadotrophin: immunological and biological studies

The alpha and beta subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 M urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The beta subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the alpha antibodies coupled to Sepharose 4B. The beta subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti HCG beta serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-beta serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-beta, hCG-alpha and hLH radioimmunoassays. While the alpha reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-alpha, the beta reactive material presenude hMG fractions was not observed in other fractions. The cross-reactivity with the anti beta serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG beta serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH ACTIVITY AFTER INCUBATION BY THE OADD test. It was observed that the anti hCG beta serum inhibits hCG more than urinary or pituitary LH.