Abstract
Since the first reports of the extraction of the lactogenic hormone by Gardner and Turner, 1 and Riddle, Bates and Dykshorn 2 involving the isoelectric precipitation of the active material from either an alkaline or acid digest of fresh or desiccated ground anterior pituitaries of sheep, cattle or hogs, little progress in purification has been reported.
The present paper presents the details of a method of further purification by which approximately a ten-fold concentration of the active principle may be effected.
The isoelectric precipitate obtained over a range of pH of 6.5 down to 5.5 from the alkaline digest is centrifuged for 20 minutes and the filtrate decanted. The precipitate is then dehydrated by repeated treatment with acetone and dried in a desiccator. It is then ground into a fine powder.
Four grams of the acetone dried powder is then thoroughly mixed with twice its volume of glacial acetic acid, centrifuged, and the brown supernatant liquid decanted into 12 times its volume of ethyl ether. This glacial acetic acid treatment is repeated 4 times or until the brown color no longer appears in the supernatant liquid. The supernatant acid from each of the extractions is decanted into the preceding acid-ether mixture. Lengthy acid treatment is to be avoided. A white flocculent precipitate containing the active principle settles out when the extraction liquids are added to the ether. The acid-ether liquid is decanted, fresh acetone is added to the precipitate and the mixture allowed to stand for 45 to 60 minutes. After centrifuging the acetone is decanted and the precipitate placed in the desiccator. After it is dry it may be ground to a fine white powder. The yield is usually about 0.4 gm. or 10% of the original precipitate. In amounts tolerated, the glacial acetic acid insoluble fraction appears to be inactive.
The active extract gives a biuret test for protein. A total of 3 mg. administered to common pigeons weighing from 300 to 325 gm. over a period of 4 days in daily injections will cause the crop glands to increase from an average of 1.5 gm. to between 1.7 and 3.8 gm. (average 2.73 gm.) showing also very distinct evidence of the proliferation of the crop gland.
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