Bennett H P, Browne C A, Solomon S
Anal Biochem. 1983 Jan;128(1):121-9. doi: 10.1016/0003-2697(83)90352-4.
The neurointermediary lobes from 190 rat pituitaries were homogenized in an acidic medium which inhibits peptidase activity and maximizes the solubilization of undamaged peptides. Octadecylsilyl-silica (ODS-silica) was used to extract the supernatant of the tissue homogenate. The ODS-silica eluate, now largely protein and salt free, was subjected to reversed-phase high-performance liquid chromatography (HPLC) employing 0.1% trifluoroacetic as counter ion. The column eluates were monitored for beta-endorphin immunoreactivity. Five immunoreactive components were observed. The most hydrophobic of these was repurified on the same HPLC column using 0.13% heptafluorobutyric acid as counter ion. Characterization of the purified peptide by gel permeation HPLC, amino acid analysis, and tryptic fragmentation indicated that it corresponded in structure to alpha-N-acetyl-beta-endorphin1-26. Amino acid analysis of the native peptide and its trypsin and carboxypeptidase fragments indicated that an alanyl residue occupies position 26. This finding is in contrast to the sequence predicted for the beta-lipotropin/corticotropin precursor by recombinant DNA techniques which suggests that the 26th residue of the beta-endorphin molecule should be valine.
从190个大鼠垂体中取出神经中间叶，在抑制肽酶活性并使未受损肽的溶解最大化的酸性介质中匀浆。使用十八烷基硅烷硅胶（ODS-硅胶）提取组织匀浆的上清液。现在基本不含蛋白质和盐分的ODS-硅胶洗脱液，采用0.1%三氟乙酸作为抗衡离子，进行反相高效液相色谱（HPLC）分析。监测柱洗脱液中的β-内啡肽免疫反应性。观察到五个免疫反应成分。其中疏水性最强的成分在同一HPLC柱上使用0.13%七氟丁酸作为抗衡离子进行再纯化。通过凝胶渗透HPLC、氨基酸分析和胰蛋白酶裂解对纯化后的肽进行表征，结果表明其结构与α-N-乙酰-β-内啡肽1-26相对应。对天然肽及其胰蛋白酶和羧肽酶片段进行氨基酸分析表明，第26位是丙氨酰残基。这一发现与重组DNA技术预测的β-促脂素/促肾上腺皮质激素前体序列相反，后者表明β-内啡肽分子的第26位残基应该是缬氨酸。
