A New Method of Labeling Human Chorionic Gonadotropin for Physiologic Studies

A new technique for labeling human chorionic gmadotropin (HCG) is described. Tritium was introduced into the molecule by reduction with tritiated borohydride after oxidation with periodate. Immunologic and biologic activity was preserved. Labeling. Oxidation of 19.6 mg of HCG in 4 ml of 0.1 M Na acetate, pH 5.6, was carried out with 50 μmoles potassium periodate at room temperature. After 30 minutes, 100 umoles glucose was added to reduce excess periodate. After dialyzing overnight in 0.1 M K2HPO4, pH 7.5, at 3 C, one-half of the preparation was reduced with 9.25 umoles of tritiated potassium borohydride (324 μCi/μmole) dissolved in 0.2 ml of 0.01 M KOH. To assure reduction of all oxidized groups, an additional 37 umoles of unlabeled borohydride was added. After adjusting the pH (5.0 to 5.5) with acetic acid, the preparation was dialyzed in cold distilled water overnight and lyophilized. All preparations were assayed by either the mouse uterine weight (MUW) or ventral prostate weight assays (VPW) (1) using 2nd International Standard HCG as reference preparation and 3×2 or 3×3 designs with 5 animals at each dose. Parallel line graded responses were used to determine relative potencies.