Use of reversed-phase and ion-exchange batch extraction in the purification of bovine pituitary peptides.
Bovine posterior pituitaries were extracted with an acidic medium designed to maximize solubilization of peptides while precipitating high-molecular-weight protein. The supernatant was then extracted with C18 reversed-phase cartridges to generate a peptide-enriched fraction. Cartridge eluates were subjected to ion-exchange extraction, using a batch procedure which fractionated the peptides into basic, acidic, and neutral pools. Amino-terminal fragments of bovine pro-opiomelanocortin were found to be resolved into separate pools by this method. The 1 to 49 fragment was eluted in the acidic pool while the 1 to 77 fragment was eluted in the basic pool. The 1 to 77 fragment was purified by reversed-phase high-performance liquid chromatography. Amino acid analysis of the fragments, generated from trypsin and V8 protease digestion of the 1 to 77 fragment, permitted assignment of cystine bridges between residues 2 and 24 and between residues 8 and 20. Results from amino sugar analysis were consistent with the presence of an O-linked oligosaccharide at threonine45 and an N-linked oligosaccharide at asparagine65.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006868:Hydrolysis; D010455:Peptides / Q000302:isolation & purification*; D010904:Pituitary Gland, Posterior / Q000032:analysis*; D011333:Pro-Opiomelanocortin / Q000302:isolation & purification; D014357:Trypsin
D010455:Peptides; D011333:Pro-Opiomelanocortin; D014357:Trypsin