An improved methodology for the extraction and partial purification of porcine hypothalamic corticotrophin releasing...

In most previous reports material with corticotrophin releasing factor (CRF) activity has been obtained from hypothalami after extraction with dilute aqueous acid. Such conditions allow substantial proteolytic degradation. By adopting conditions designed to precipitate proteases and by using information on the nature of CRF gained from earlier studies, rapid large scale extraction and partial purification of porcine hypothalamic CRF in high yield was achieved. After extraction with 0.2 m-HCl; acetone (1:1, v/v), centrifugation and ultrafiltration, considerable preliminary purification of the CRF activity was achieved by adsorption onto carboxymethylcellulose and subsequent elution at increased salt concentration. Following ion-exchange chromatography of the extract on carboxymethylcellulose, CRF activity was obtained in good yield (minimal effective dose of about 1-2 micrograms/ml) for ACTH release in an in-vitro CRF bioassay utilizing a coupled isolated pituitary cell-adrenal cell system. The data indicated that the previously reported heterogeneous corticotrophin releasing factors of low activity may be a consequence of proteolytic degradation.