Serum gonadotrophin

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Abstract
Improvements have been made in the method previously published for the preparation of gonadotrophic extracts from pregnant mares'' serum. By the improved method, fractions are obtained assaying 200-1000 i.u./mg. and representing a yield of approx. 70% of the original activity of the serum. Further purification of the product is accomplished by (a) shaking the dry solid with 50% (v/v) ethanol containing buffer pH 4.8, (b) removal of material insoluble in 56% (v/v) ethanol at pH 6.5 and finally (c) fractionation by ethanol at pH 1.5. In this manner preps. have been obtained assaying 12,500 i.u./mg. Normal horse serum, similarly treated, yielded only a trace of material devoid of gonadotrophic activity, but containing approx. the same amt. of carbohydrate as the most active gonadotrophic preps. Carbohydrate content is no measure of gonadotrophic activity. The duration of contact of material, as in the ultimate stage, with aqueous acid soln., pH 2.5, does not affect the potency of the resulting product, whence it is concluded that the action of the acid is not a progressive cleavage of inert material from an active moiety such as a prosthetic group. Studies on the stability of active fractions show that: (a) 85% of their activity is lost by heating at 60[degree] for 96 hr., (b) the rate of inactivation is the same in the presence of air, O2 or N2, (c) spontaneous inactivation occurs in watery soln. at 37[degree] and more rapidly in the presence of acid or alkaline buffers; the pH range 3.5-9.5 was studied. Inactivated material could not be reactivated by quinol. The hexose: hexosamine ratio of the purified material was found to be 2:1, similar to that of other protein fractions of normal serum. The possible relationship between serum gonadotrophin and other sero-glycoproteins is discussed.

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