Preparation of Pituitary Adrenotropic Hormone. ∗

Abstract

This is a preliminary description of a relatively simple procedure devised for the isolation of purified and highly active preparations of adrenotropic hormone from whole hog pituitary glands. The extraction and fractionation procedures consist of the following steps: (1) Extraction and isolation of a fraction designated as crude prolactin by the procedure originally developed by Lyons 1 and adopted by White, Bonsnes and Long 2 as the first step for the subsequent preparation of crystalline prolactin. This “crude prolactin” contains adrenotropic hormone activity. (2) Solution of the crude prolactin preparation in 2% concentration at pH 9.0 and removal of precipitates which form by careful adjustment of pH to 8.0, 6.6 and 5.4. (3) Supernatant obtained in Step 2, after removal of pH 5.4 insoluble material (prolactin), is freed from traces of prolactin by the addition of 7 ml of saturated (NH₄)₂SO₄ to each 100 ml of solution. The precipitate is separated and discarded. (4) Acetone is added to the supernatant to a concentration of 80%. The adrenotropic fraction precipitates. (5) This precipitate is dissolved in water (volume used being two-thirds of that employed in Step 2) and the solution mixed with one-half its volume of concentrated NH₄OH. The ammoniacal solution is allowed to stand at room temperature for 7 hours. (6) Acetone is then added to above solution to a concentration of 90%. (7) The precipitate is dissolved in one-third volume of water used in Step 2, and dialyzed to remove salt. (8) The adrenotropic fraction is precipitated by lowering of pH to 4.7. The precipitate may be dried by washing 3 times with acetone at the centrifuge and finally drying in vacuo over concentrated sulfuric acid. The yield of dry, purified adrenotropic fraction has been of the order of 500 mg from each 1000 g of whole hog pituitary glands.