title: Separation and isolation of human apolipoproteins C-II, C-III0, C-III1, and C-III2 by chromatofocusing on the Fast Protein Liquid Chromatography system.
abstract: Chromatofocusing, which separates proteins based on differences in isoelectric point, has been used on the Fast Protein Liquid Chromatography (FPLC) system (Pharmacia) to separate the C apolipoproteins from human very low density lipoproteins (VLDL). Using a Mono P column (Pharmacia), a pH gradient between pH 6.2 and pH 4.0 was generated using buffers containing 6 M urea, at a flow rate of 0.5 ml/min. Typically, runs took approximately 45 min. Chromatofocusing of delipidated whole VLDL produced sharp, well-resolved peaks for the C apolipoproteins. However, as determined by analytical isoelectric focusing (IEF), the apolipoprotein E isoforms were not separated from apoC-II, and they contaminated the other apoC species to a variable extent. In addition, apoC-II was not resolved from apoC-III0. Preliminary precipitation of VLDL with acetone prior to delipidation removed both apolipoproteins E and B. Using a start buffer of 25 mM histidine, pH 6.2, and a 1:30 dilution of the polybuffer exchanger (eluting buffer), apoC-II, C-III0, C-III1, and C-III2 were well resolved in run-times of approximately 60 min. The C apoproteins proved to be pure by analytical IEF and immunoassay with monospecific antisera against apoC-II and C-III. Recovery was over 90% of the protein chromatographed. In addition, a variant of apoC-II present in VLDL of a hypertriglyceridemic subject was clearly resolved from the other C apolipoproteins. This technique is superior to conventional methodology in terms of its time saving and high resolution. The application of this technique to the study of C apolipoprotein variants and C apolipoprotein specific radioactivity determinations is possible.
authors: Department of Medicine, University Hospital, University of Western Ontario, London, Canada.
authors: M W
authors: MW
authors: Huff
authors: W L
authors: WL
authors: Strong
mesh_terms: D000096:Acetone; D053304:Apolipoprotein C-II; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D006801:Humans; D007526:Isoelectric Point; D008079:Lipoproteins, VLDL / Q000032:analysis*; D012995:Solubility
chemical_list: D053304:Apolipoprotein C-II; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D008079:Lipoproteins, VLDL; D000096:Acetone