Occurrence of Fragmentation of Free and Combined Forms of the β-Subunit of Human Chorionic Gonadotropin*

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Abstract
In an attempt to further study various fragments of free and combined forms of hCG.beta. present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel elecrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG.beta.(111-116) portion (monoclonal antibody FB12) antiserum to the hCG.beta.(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG.beta. nicked subunit (.beta.NS) [hCG.beta.-(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 immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG.beta.-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the .beta.NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free .beta.-subunit contained .beta.NS. Furthermore, free hCG.beta. 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 .beta.-subunit by a two-step chromatography procedure, identification of hCG ad hCG.beta. 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 .beta.NS in sera of four patients with choriocarcinoma tumors. In those sera, hCP appeared to be nicked. This study demonstrates that the .beta.-subunit of hCG is modified by multiple fragmentations.

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