Application of reversed-phase high-performance liquid chromatography to the separation of apolipoproteins A-IV, A-I and E from rat high-density lipoprotein.

Apolipoproteins A-IV, A-I and E from rat high-density lipoprotein (HDL) were successfully purified by reversed-phase high-performance liquid chromatography (RP-HPLC), using a method which we have previously developed for the separation of apolipoproteins A-IV, A-I and E from human lymph chylomicrons [T. Tetaz, E. Kecorius, B. Grego and N. Fidge, J. Chromatogr., 511 (1990) 147]. Since analytical-scale RP-HPLC indicated that the C apolipoproteins from rat HDL coeluted with both apo A-IV and apo A-I, delipidated rat HDL was first subjected to preparative-scale size-exclusion HPLC (HPSEC) on a Serva Si300 column, which effectively separated the C apolipoproteins from all but apolipoprotein E. Fractions from HPSEC which were enriched for apolipoproteins A-IV, A-I or E were directly applied to RP-HPLC on a TSK Phenyl-5PW column. This procedure yielded fractions containing apolipoproteins A-IV, A-I or E which were pure as assessed by N-terminal sequencing and silver staining of sodium dodecyl sulphate-polyacrylamide gels.