James S, Bennett H P
J Chromatogr. 1985 Jun 19;326:329-38. doi: 10.1016/s0021-9673(01)87458-6.
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.
用一种酸性介质提取牛垂体后叶，该酸性介质旨在使肽的溶解最大化，同时沉淀高分子量蛋白质。然后用上样于C18反相柱进行提取，以产生富含肽的级分。柱洗脱液采用分批法进行离子交换提取，将肽分为碱性、酸性和中性组分。发现通过该方法可将牛阿黑皮素原的氨基末端片段分离到不同组分中。1至49片段在酸性组分中洗脱，而1至77片段在碱性组分中洗脱。通过反相高效液相色谱法纯化1至77片段。对1至77片段经胰蛋白酶和V8蛋白酶消化产生的片段进行氨基酸分析，确定了2位和24位残基之间以及8位和20位残基之间的胱氨酸桥。氨基糖分析结果与苏氨酸45处存在O-连接寡糖和天冬酰胺65处存在N-连接寡糖一致。
