Weinberg R, Patton C, DaGue B
Department of Medicine, University of Texas Health Science Center, Houston 77225.
J Lipid Res. 1988 Jun;29(6):819-24.
We have developed a rapid reverse phase high pressure liquid chromatographic technique capable of separating apolipoproteins A-I, A-II, and A-IV and their constituent isoforms. The separations were performed on a 30 cm x 3.9 mm C18 reverse-phase silica-based column (Waters Associates) using a 47-55% gradient of acetonitrile in 0.1% trifluoroacetic acid. Injection of a mixture of equal weights of apolipoproteins A-I, A-II, and A-IV yielded a single peak for apoA-IV, two peaks of apoA-I isoforms, and three peaks of apoA-II isoforms, with negligible overlap of each peak. When applied to the isolation of apoA-IV from a crude protein mixture obtained by incubation of phospholipid-triglyceride emulsion particles with lipoprotein-depleted plasma, the technique yielded a single peak of highly pure apoA-IV cleanly separated from isoforms of apoA-I and higher molecular weight proteins. A positive linear correlation was observed between apoprotein hydrophobicity and column retention time, thus indicating that the system provided a true reverse phase separation. We conclude that reverse phase high pressure liquid chromatography can separate human apolipoprotein A-IV from isoforms of apoA-I and apoA-II on the basis of differences in mean molecular hydrophobicity. The technique has direct application to the isolation of human apolipoprotein A-IV.
我们开发了一种快速反相高压液相色谱技术，能够分离载脂蛋白A-I、A-II和A-IV及其组成亚型。分离在一根30 cm×3.9 mm的C18反相硅胶柱（沃特斯公司）上进行，使用在0.1%三氟乙酸中的47%-55%乙腈梯度。注入等重量的载脂蛋白A-I、A-II和A-IV的混合物，得到载脂蛋白A-IV的一个单峰、载脂蛋白A-I亚型的两个峰和载脂蛋白A-II亚型的三个峰，每个峰的重叠可忽略不计。当应用于从通过将磷脂-甘油三酯乳液颗粒与脱脂血浆孵育获得的粗蛋白混合物中分离载脂蛋白A-IV时，该技术产生了一个高度纯的载脂蛋白A-IV单峰，与载脂蛋白A-I亚型和高分子量蛋白干净地分离。观察到载脂蛋白疏水性与柱保留时间之间呈正线性相关，因此表明该系统提供了真正的反相分离。我们得出结论，反相高压液相色谱可以根据平均分子疏水性的差异，将人载脂蛋白A-IV与载脂蛋白A-I和A-II的亚型分离。该技术可直接应用于人载脂蛋白A-IV的分离。
