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.

A Comparison of Procedures for the Isolation of Adrenocorticotrophin From Human Pituitary Glands

“…Optimal yields of intact hormone can only be achieved if proteolysis is minimized before extraction. Substantial losses of ACTH may occur in pituitary glands collected at routine autopsies since degradation in intact pituitaries occurs within hours at room temperature (Hussa et al 1969).…”

“…Oxycellulose (10-12% carboxyl), carboxymethylcellulose (Whatman CM-23 and CM-32), Sephadex G-50 (fine grade) and Amberlite CG-50 (IRC-50, 200-400 mesh) were obtained and prepared as described by Hussa, Winnick & Landon (1969). Amberlite CG-50 was pretreated by a modification of the method of Hirs, Moore & Stein (1953) in which precycling through the sodium and hydrogen forms was per¬ formed at 100°C for 24 h. Porous glass beads (Spherosil, type A, 100-200 µ ) were kindly supplied by K. W. Chemicals Ltd, 55-57 High Holborn, London W.C. 1.…”

“…The time of adsorption of crude ACTH extract to oxycellulose was reduced to 2 h from the 24 h used by Hussa et al (1969), since 90 % of the total ACTH was adsorbed by this time. The oxycellulose step achieved a 28-fold purification as measured by immunoassay with only an 8% loss of N-terminal reactive hormone.…”

“…The excellent purification and small losses are probably related to the brief adsorption period and the use of rela¬ tively large amounts of oxycellulose (50 % by weight of crude extract adsorbed). The oxycellulose-purified powder was chromatographed on a large CM-23 column (2 60 cm) in order to improve the resolution obtained by Hussa et al (1969). Four fractions were obtained (Text- fig.…”

Purification and Characterization of Porcine Corticotrophin-Like Intermediate Lobe Peptide

“…Extraction of peptides Several procedures, based on those previously published for ACTH (Hussa, Winnick & Landon, 1969) were compared by measuring the yields of immunoactive ACTH from 1 g portions of powder extracted by the different procedures. All mani¬ pulations were carried out at 4°C.…”