THE DEACTIVATION OF HCG BY NICKING AND DISSOCIATION

Human chorionic gonadotropin (hCG) is composed of an alpha- and a beta-subunit, joined noncovalently. A proportion of hCG molecules in pregnancy serum and urine samples have nicks or a missing peptide linkage between either beta-subunit residues 44 and 45 or beta-subunit residues 47 and 48. These nicks ablate the steroidogenic activity of hCG. We examined the source of nicking, and the occurrence and stability of nicked hCG molecules produced during pregnancy. We investigated the source of nicking. Standard hCG was added to three samples of whole blood, and incubated 18 h at 37 C. No change in extent of nicking was detected. However, nicking of hCG beta-subunit was detected by gel electrophoresis (bands at M(r) = 17,000 and M(r) = 22,000, corresponding to the peptides beta1-47(44) and beta48(45)-145, respectively) in culture fluids from first trimester placental explants and from JAr malignant trophoblast cells. We inferred that nicking occurs before or immediately upon secretion by trophoblast tissue. We examined the occurrence of nicking. Levels of total hCG (nicked + nonnicked) and intact hCG (nonnicked) were determined in 233 serum and 168 urine samples from 4-40 weeks of pregnancy. From the two measurements the extent of nicking was estimated. A linear relationship was indicated between advancing weeks of gestation and increasing extent of nicking (regression analysis, months vs. percent nicked, 95% correlation). Minimum nicking was observed in serum from the first 2 months of pregnancy (mean = 9% of hCG molecules), increased nicking in the months after, and maximum nicking in samples from the last 2 months of pregnancy (mean = 21% of hCG molecules, t test first 2 months vs. last 2 months, P < 0.00005). Similar results were observed with urine samples (first 2 months mean = 9%, last 2 months = 27%, t test P < 0.00005). We concluded that nicking is more prevalent after the hCG peak (after 2 months of pregnancy). Finally, we examined the stabilities of nicked and intact hCG molecules. Standard hCG (batch CR127, 20% nicked) and hCG preparation C5 (100% nicked) were incubated for varying times in whole blood. C5 hCG dissociated rapidly into free alpha- and beta-subunits (dissociation half-life 22 +/- 5.2 h), over 30 times faster than standard hCG (dissociation half-life 700 h). We inferred that nicked hCG rapidly dissociates, and that the relative amount of nicked molecules produced by the trophoblast may, considering circulating (37-41 h) and dissociation half-lives, be 3.4-3.6 times higher than that measured in serum samples. If nicked molecules rapidly dissociate then levels of free subunits should rise as the extent of nicking increases. From the first to the last month of pregnancy, extents of nicking rise from 8-31% of hCG molecules, urine free a-levels, free beta-subunit, and beta-core fragment (degradation product of beta-subunit) levels rise by similar proportions, from 76-360%,18-99%, and from 58-305% of hCG levels. hCG functions in the first 2 months of pregnancy to maintain progesterone production by the corpus luteum. After this period the hormone has no known function. The findings presented here indicate that nicking or deactivation of hCG occurs in or around trophoblast tissue, that nicking is more prevalent after the first 2 months of pregnancy when hCG has no known function, and that once nicked, hCG rapidly dissociates into free alpha- and beta-subunits.