Gonadotrophins were prepared from human postmenopausal urine with the kaolin-acetone method and purified by adsorption of impurities on diethylamine ethyl cellulose (DEAE-C). The last step, namely chromatography on the column of permutit, resulted in a significantly improved specific activity. The immunoelectrophoresis showed that the purified HMG is not homogeneous and that the constituents are glycoproteins which migrate into the region α1, α2 and β globulins.

A new approach to the preparation of biologically pure FSH from HMG is described. The method entails selective binding of the LH to antibodies against HCG. The bound LH was separated from FSH by DEAE-C chromatography. An FSH preparation was obtained with a potency of 329.7 IU-FSH/mg and undetectable LH activity at the highest dose tested. This preparation, however, was not homogeneous in the immuno-electrophoretic analysis. The nature of the LH activity »unseparable« from FSH by physico-chemical procedures is discussed.

The response of the uterine weight of immature mice to FSH and LH preparations with low contamination by each other was studied. The new approach to the biological assay of LH, which could be designated the »uterine-augmentation« test, was based on injecting intact immature mice with a constant dose (4.44 IU) of a biologically pure FSH preparation plus increasing amounts of extracts containing LH. A dose as little as 0.068 IU of LH is capable to increase the uterine weight when injected together with 4.44 IU of biologically pure FSH. A significant uterine response can be obtained when animals are injected with 29.4 μg of 2nd IRP-HMG (equivalent to 0.128 IU-LH and 0.128 IU-FSH). When a constant dose of 4.44 IU of biologically pure FSH is added to each dose level of the 2nd IRP-HMG, a significant uterine response is obtained with 14.7 μg of this reference preparation. The end point was the mouse uterine weight. Some determinations of the LH activity in urinary extracts have been performed according to this new method and from the results it seems that the LH activities, as determined by the ovarian ascorbic acid depletion method, agree with those determined by the uterine-augmentation test. It also seems that the uterine weight response is proportional to the LH activity and not dependent on the FSH-LH ratio of the samples assayed.

The further purification of HMG obtained from postmenopausal urine with kaolin-acetone, DEAE-C and permutit chromatography was performed using gel filtration on Sephadex (G 200). The potency of the purest fraction isolated was about 2.5 times higher than that of the starting material. The total recovery of biological activity was 96 %.Several methods using ion-exchange materials for the purification of human pituitary gonadotrophin (HPG) and human menopausal gonadotrophin (HMG) have been proposed.The chromatography of HPG on Sephadex (G 75) and DEAE-Sephadex (A 50) were attempted by Beltendorf et al. (1962).The present paper deals with the further purification, using gel filtration on Sephadex (G 200) of HMG obtained from postmenopausal urine with kaolinacetone, DEAE-cellulose and permutit chromatography. MATERIALS AND METHODSAs starting material for the further purification with gel filtration on Sephadex, HMG Pergonal-26 E43 C was used. This was obtained by Donini el al. (1963) by means of the kaolin-acetone method, followed by 10°/o ammonium acetate in 70% ethanol by DEAE-C purification and, finally, by permutit chromatography.The gel filtration was performed on Sephadex (G 200, 140-400 mesh.). Dry Sephadex