Masler E P, Hagedorn H H, Petzel D H, Borkovec A B
Life Sci. 1983 Nov 7;33(19):1925-31. doi: 10.1016/0024-3205(83)90677-x.
We report the use of reverse-phase liquid chromatographic techniques for the isolation of a steroidogenic neuropeptide (EDNH) from mosquito heads. Activity of fractions was assayed by measuring the ability of ovaries to produce ecdysteroid in vitro. Dose response profiles using crude head extracts or partially purified EDNH were nearly identical, indicating that the methods of preparation did not alter biological activity. EDNH activity eluted from a reverse-phase HPLC (RP-HPLC) column primarily near 35 percent acetonitrile using a linear gradient. Methods developed with an analytical RP-HPLC column were successfully adapted for preparative work. Active fractions from the preparative RP-HPLC were further purified on a second analytical column under isocratic conditions at 30% acetonitrile. Two adjacent UV absorbing peaks were found, each with EDNH activity. Activity was sensitive to proteolysis.
我们报告了使用反相液相色谱技术从蚊子头部分离一种促类固醇生成神经肽（EDNH）的方法。通过测量卵巢在体外产生蜕皮甾体的能力来测定各馏分的活性。使用粗头提取物或部分纯化的EDNH的剂量反应曲线几乎相同，这表明制备方法没有改变生物活性。使用线性梯度时，EDNH活性主要在反相高效液相色谱（RP-HPLC）柱上约35%乙腈处洗脱。用分析型RP-HPLC柱开发的方法成功地应用于制备工作。制备型RP-HPLC的活性馏分在第二个分析柱上于30%乙腈等度条件下进一步纯化。发现了两个相邻的紫外吸收峰，每个峰都具有EDNH活性。该活性对蛋白水解敏感。
