Studies on the Separation and the Extraction of Anterior Pituitary Hormone by Paper-electrophoresis

Abstract

Aceton dry powder of pig's anterior hypophysis were extracted with 2% saline water, and were centrifuged. The supernatant fluid was fractionated by continuous paper-electrophoresis (Condition : 26mA/35cm. 500v/40cm, ionic strength 0.05, Phosphoric buffer pH 8.5).Each fraction was biologically assayed ; TSH activity by Greenspan's method, Gonadotropic activity by Florsheim's method, STH activity by Tibia-Test, and ACTH activity by Sayers-Munson's method. The fraction in the middle, TSH activity was clearly detected with slight gonadotropic activity. Other activities were not accounted. Contents of the fraction, in which TSH activity was revealed, were ultracentrifuged. The sedimentation curve was monophasic and the sedimentation constants was 2.28. Thus the molecular weight was calculated as about 20000.In phosphate buffer solution, pH 4.5, the extracted substances of aceton dry powder of pig's anterior hypophysis were separated by continuous paper-electrophoresis into six fractions. In this case, as it was anticipated on account of acid solution thyrotropic activity would be diminished and only gonadotropic activity would be remained, the gonadotropic activity was measured by weighing the ovaries, uterus, prostate and seminal vesicle of hypophysectomized immature rats and the ovaries were examined histologically. On the second and the third fraction, which are numbered from anod, gonadotropic activity was found. The LH action was recognized in the second fraction and the FSH action in the third fraction.NIH-FSH and NIH-LH which were introduced by Dr. Wilhelmi were also separeted by paperelectrophoresis, then NIH-FSH is separeted into two fraction, but NIH-LH separeted into only one fraction, so the purity of NIH-LH was confirmed.Anteron was injected into hypophysectomized rat intravenously, ten minutes later blood was taken. The serum separeted by continuous paperelectrophoresis were examined for gonadotropic activity of each fraction by the method of weighing mouse uterus.On gamma-globulin in the serum of rat, gonadotropic activity was recognized.Crude gonadotropic substances of pregnant woman's urine and postmenopausal urine obtained by kaolin absorption method, were separeted into some fraction by continuous paperelectrophoresis (Condition : 26mA/35cm, 450v/40cm, ionic strength 0.05, phosphate buffer solution, pH 4.5).Crude substance of postmenopasal woman's urine was separated into 3 fractions. Only in the first fraction, gonadotropic activity was revealed ; its FSH-action was very strong, but its LH-action was very weak.Crude substance of the pregnant woman's urine in the former half term was separated into four fractions. Gonadotropic activity was recognized in the first and the second fraction ; The LH-action of it in the second fraction was stronger than that of the first fraction and FSH-action in the first fraction was stronger than that of the second.Crude substance of the pregnant woman's urine in the later half term was separated into three fractions. In the first and second fraction of it gonadotropic activities were found, but FSH-action was a little weak comparing with that of the gonadotropic activity of the urine in the former half term. Even in the case of human chorionic gonadotropin and postmenopausal gonadotropin, LH-action and FSH-action were not separated perfectly.1) Differences by paperelectrophoric condition.TSH activity exists only in the middle fraction fractionated in basic solvent, but in acid solvent thyrotropic activity is apt to be lost. On the other hand, gonadotropic activity was not lost even in acetic solvent and in basic solvent. Therefore, for separation, extraction and purification of TSH, any procedure under acetic solvent are not desirable.2) Thyrotropic substances and gonadotropic substances in serum.Both TSH activity and gonadotropic activity were recognized on gamma-globulin.