The small-scale isolation and characterization of adrenocorticotrophin

Abstract

Several methods for isolating adrenocorticotrophin from small quantities of porcine and bovine pituitary tissue are compared. Initial extraction of the hormone by an acid–acetone technique was simpler and more efficient than one employing acetic acid extraction and ether precipitation. Subsequent purification procedures utilizing adsorption of the peptide on to oxycellulose realized the highest yields. CM-cellulose-column chromatography followed by Sephadex-gel filtration were suitable final steps for obtaining highly purified adrenocorticotrophin. The purity of the hormone was demonstrated by determining its amino acid composition, C-terminal analysis, polyacrylamide-gel electrophoresis, chymotrypsin digestion and paper electrophoresis and by radioimmunoassay and bioassay. Adrenocorticotrophin was found to be rapidly destroyed in intact and especially in homogenized glands kept at room temperature. At 4° the rate of destruction was less rapid and at −20° losses were minimal.