RECENT STUDIES ON THE PROBLEMS INVOLVED IN THE PREPARATION, PROPERTIES AND BIOASSAY OF ADRENOCORTICOTROPHIC HORMONE

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Abstract
A review is presented of differences in the relative potency of various ACTH prepns. when detd. by different tests (ascorbic acid depletion vs. adrenal wt. maintenance vs. adrenal cholesterol depletion vs. lymphocyte or thymocyte destruction vs. eosinophil cell depression). Variations in response may indicate presence of several active components in ACTH, or may reflect differences in rate of absorption from injn. depots. Li-Sayers ACTH protein is shown not to be homogenous by electro-dialysis, paper electrophoresis, 25-transfer counter-current distr. and oxycellulose adsorption. These methods achieve 4 times the concn. of activity over activity of original prepn. and show several components in the Li-Sayers purified ACTH prepn. A 10-20 times concn. of ACTH activity/mg. N can be achieved by peptic digestion, followed by displacement chromatography on a 1:9 charcoal: infusorial earth column with increasingly long chain n-alcohols, starting with 1% n-octyl alcohol dissolved in 50% ethanol containing 0.1 [image] HC1. Using 2% n-decanol in 50% ethanol-0.1 [image] HCl, 70% of N appears in the 1st 40 ml., but this is inactive; and most of the ACTH appears in the 50-55 ml. fraction at a potency of 20-40 USP units/mg. All of the N and ACTH were accounted for in this expt. A method is given for fractionating acid-acetone powder from sheep pituitaries, precipitation of impurities with NaCl at pH 3.0 (20% of total solids appear in this fraction), followed by oxycellulose adsorption (after Astwood). The final yield was about 1% of original wt. of powder, with 50-100 USP units/mg. This prepn. contains 16.1% N, 0.7% S, 3% tryptophan, 2.7% methionine, 12.6% arginine, 13.2% lysine, 7.5% aspartic acid, 15.4% glutamic acid and no histidine. The molecular wt. is about 10,000 (ultracentri-fuge and cellulose dialysis). The isoelectric point is about pH 9. By peptic digestion, potency was raised to 52 USP units/mg., and by acid hydrolysis (0.2 M HCL, 100[degree]C, 3 hrs.) to about 270 USP units/mg. The protein can also be purified by trichloroacetic acid fractionation followed by displacement chromatography. No correlation could be found between ACTH activity of various prepns. and melanophore activity.
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