OCCURRENCE OF FRAGMENTATION OF FREE AND COMBINED FORMS OF THE BETA-SUBUNIT OF HUMAN CHORIONIC-GONADOTROPIN

In an attempt to further study various fragments of free and combined forms of hCGβ present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCGβ-(111-116) portion (monoclonal antibody FB12) antiserum to the hCGβ (8-16) portion or antiserum which was specific for fragments ending at residue 47.Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCGβ nicked subunit (ONS) [hCGβ-(48-145)], three other fragments of mol wt 18, 000 (F1), 16, 500 (F2), and 12, 000 (F3) were detectable after cleavage of disulfide bonds. Both the immune reactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCGβ-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the βNS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free β-subunit contained βNS. Furthermore, free hCGβ present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its β-subunit by a twostep chromatography procedure, identification of hCG and hCGβ immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of βNS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the β-subunit of hCG is modified by multiple fragmentations. © 1990 by The Endocrine Society.