The role of
carbohydrate in the heterogeneity of hCG and its subunits is unclear. To study this question, we chromatographed over
Sephadex G-100 an extract of term placenta as well as sera from a woman in the first and third trimesters of pregnancy and sera from two patients with nontrophoblastic
malignancies. Samples were cochromatographed with radiolabeled urinary standards. hCG from first trimester pregnancy serum showed multiple peaks on G-100. The dominant peak eluted with an apparent molecular weight (72,000) higher than that of hCG from third trimester serum (63,000), urine (6),000), and placenta (59,000). hCG from both
malignancy sera eluted with an apparent molecular weight (62,000) similar to that of hCG from third trimester and urinary hCG. Free
hCG alpha from all sera eluted with a similar apparent molecular weight (29,000), which was higher than that of placental and urinary free alpha-subunit (22,000) and the alpha-subunit dissociated from intact hCG from all sources (22,000--23,000). The subunits were dissociated in the denaturing medium of 6 M
guanidine-HCl, pH 3.0, and chromatographed in this medium over
Sepharose CL-6B. This eliminated all of the differences in apparent molecular weight among corresponding forms that were found on G-100. All forms of
hCG alpha coeluted with a chemically identified 80%
deglycosylated hCG alpha. hCG and free alpha-subunits were incubated with mixed
exoglycosidases which lacked detectable
protease activity and were then rechromatographed on G-100. After deglycosylation, hCG from different sources eluted with a considerable heterogeneity (mol wt range, 40,000--50,000) not present in the native forms. Despite the heterogeneity of native free alpha-subunit from various sources, deglycosylation produced a common species with apparent molecular weights of 11,000--12,000, close to the chemically determined molecular weight of the
polypeptide chain (10,400). These studies suggest that1) ectopic serum hCG and free alpha-subunit are similar to corresponding eutopic forms; 2) serum hCG and free alpha-subunit from all sources are more glycosylated than placental or urinary forms; 3) first trimester hCG is more glycosylated than other forms of hCG; and 4) serum free alpha-subunit is more glycosylated than the alpha-subunit which combines with
hCG beta to form intact hCG.