Journal of Lipid Research, Vol 29, 819-824, Copyright © 1988 by Lipid Research, Inc.
Analytic and preparative separation of apolipoproteins A-I, A-II, and A- IV by reverse phase high pressure liquid chromatography
R Weinberg, C Patton and B DaGue
Department of Medicine, University of Texas Health Science Center, Houston 77225.
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.
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