Abstract
Human choriogonadotropin was isolated from urine of patients with hydatidiform mole by acid and salt precipitation, immunoaffinity, and DEAE-Sephadex chromatography. Polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and NH2-terminal amino acid analysis showed that the product obtained is essentially homogeneous. This choriogonadotropin was found to resemble the choriogonadotropin from urine of normal pregnant women in amino acid composition but to differ from it in having a lower content of N-acetylglucosamine and mannose.
Highlights
Human choriogonadotropin was isolated from urine of patients with hydatidiform mole by acid and salt precipitation, immunoafflnity, and DEAE-Sephadex chromatography
When 2000 IU of crude hCG-hydatidiform mole were charged onto an anti-hCG IgG Sepharose column (0.8 x 1.2 cm) and washed with NaCl/Pi, the bulk of the nonspecific proteins passed through the column unadsorbed
When the above immunologically active fraction eluted by 2 M sodium trichloroacetate was subjected to DEAE-Sepharose chromatography, the elution pattern shown in Fig. 2 was obtained
Summary
The precipitate containing the bulk of the gonadotropic activity was collected by centrifugation and dissolved in about 100 ml of distilled water. This protein solution was dialyzed against distilled water for 2 days. The two fractions eluted by 1 M sodium trichloroacetate and 2 M sodium trichloroacetate were diluted respectively with distilled water Each of these fractions was dialyzed against several changes of 0.1 M ammonium bicarbonate for 2 days. Hexosamines were released by hydrolysis of the sample with 2 N HCl at IlO’C for 5 h Their derived alditol acetates- were again analyzed by gas-liq;lib chromatography using a column packed with 3% poly(A) 103 on Gaschrom Q (100/120 mesh) at 230°C. Sialic acid was assayed by the thiobarbituric method of Warren after the sample was hydrolyzed with 0.05 N sulfuric acid at 80°C for 1 h [34]
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