Elution of lipoprotein fractions containing apolipoproteins E and A-I in size exclusion on Superose 6 columns is sensitive to mobile phase pH and ionic strength.
Separation of lipoproteins secreted from McA-RH7777 (rat hepatoma) cells by Superose 6 column size-exclusion chromatography, using PBS buffer (NaCl 150 mM, sodium phosphate 10 mM, pH 7.5, EDTA 1 mM), produced apolipoprotein (apo) E or A-I profiles that did not correlate with lipoproteins separated by density ultracentrifugation. By density ultracentrifugation, apoE and apoA-I were mostly (> 90%) confined to high-density lipoproteins (HDL, d = 1.063-1.023 g/ml), but by chromatography apoE and apoA-I were recovered in all lipoprotein classes, including low-density lipoproteins (LDL), HDL, and post-HDL. Moreover, the elution volume of phenol red on Superose 6 greatly exceeded the total column volume. These discrepancies were attributable to pH and ionic strength effects. In low ionic strength, high pH buffer (Tris 25 mM, pH 8.3), elution volumes of lipoproteins, albumin, and phenol red were minimized. Elution volumes increased 25-70% when buffer pH was lowered at constant ionic strength (Tris 25 mM, pH 7.4) or when ionic strength was increased at constant pH (Tris 25 mM, pH 8.3, NaCl 500 mM). Altered phase partition appeared to cause the altered elution volumes, since recovery (measured as analyte peak area), resolution (measured as peak width at half height), and column void volume varied little from buffer to buffer. In Superose 6 size-exclusion chromatography with PBS buffer, then, elution volumes vary with pH and ionic strength. We propose that TBE buffer (Tris-borate 89 mM, pH 8.3, EDTA 2 mM) may produce fewer artefacts than PBS. With TBE there were (i) better correlation between size-exclusion and ultracentrifugal fractions, (ii) lower elution volumes, and (iii) less ¿smearing¿ of McA-RH7777 apoE and apoA-I containing lipoprotein bands.
Westerlund
J
J
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Yao
Z
Z
D000818:Animals; D016632:Apolipoprotein A-I / Q000032:analysis / Q000302:isolation & purification*; D001057:Apolipoproteins E / Q000032:analysis / Q000302:isolation & purification*; D002021:Buffers; D002847:Chromatography, Agarose; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008074:Lipoproteins / Q000737:chemistry / Q000302:isolation & purification*; D009994:Osmolar Concentration; D051381:Rats; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D002021:Buffers; D008074:Lipoproteins