Preparation of Gonadotropic Substance by Tungstic Acid Precipitation

Abstract

We presented data on the quantitative precipitation of small quantities of prolan with tungstic acid. 1 Extending our work to pregnancy urine we found that tungstic acid would produce a precipitate which contained most of the gonadotropic substance and a considerable quantity of both protein and tungstic acid. Since our interest lay in the purification of the gonadotropic material, several processes for the removal of the impurities wrere studied. The barium precipitation used in our earlier work and which was also used for the decomposition of the phosphotungstate precipitate (Wiesner and Marshall, 2 Zondek, Scheibler and Krabbe 3 proved to be less satisfactory than the treatment with certain inexpensive alkaloids.

To one liter of urine add 20 cc. of sodium tungstate (10%). Acidify this mixture to congo red paper with sulfuric acid. Centrifuge and wash the precipitate once with acetone. A little distilled water and some solid brucine are added, the mixture thoroughly stirred and then centrifuged. The clear supernatant solution contains nearly all of the gonadotropic substance of the original urine.

Although our experience is somewhat limited it seems probable that sodium molybdate may be used instead of sodium tungstate. Quinine and possibly benzidine hydrochloride or 8, hydroxy-quin-oline may be used in place of the brucine. Barium hydroxide gives less satisfactory preparations and sodium hydroxide or aqueous pyridine dissolves nearly all of the tungstate precipitate.

Since we have found that some purified preparations of the gonadotropic substance fail to give a precipitate with tungstic acid, it is probable that the active material is carried down by adsorption. The function of the brucine is 2-fold; to release the gonadotropic material from the precipitate and to form an insoluble tungstate which prevents the tungsten from dissolving. As shown in the table, the tungstic acid procedure gives much higher yields than those obtained by the benzoic acid method (Katzman and Doisy 4 ). The yields obtained by the use of brucine or quinine compare favorably with those of the crude sodium hydroxide solutions of the tungstate precipitate.