Analytic and preparative separation of apolipoproteins A-I, A-II, and A-IV by reverse phase high pressure liquid chromatography

Abstract

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.