Chromatographic techniques for the isolation and purification of lipoproteins.

Various modes of chromatography are available for lipoprotein separation. Gel permeation and affinity chromatography are used for preparative purposes and to separate lipoproteins according to size and apolipoprotein content, respectively. Development of rigid supports for gel permeation has led to large improvements in speed and resolution. Reversed-phase high-performance liquid chromatography (HPLC) of apolipoproteins offers the best performance in terms of speed and resolution of structural variants. Due to its high speed and superior resolving power, the recently developed technique of capillary electrophoresis should emerge as an important method for lipoprotein analysis.

Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries.

The addition of sodium dodecyl sulfate (SDS) micelles in the running buffer can be used to resolve mono- and diphosphorylated isomers of the insulin receptor peptide by capillary electrophoresis. The effect of SDS on peptide resolution is very dependent on pH. Complete resolution of three monophosphorylated isomers is achieved in uncoated capillaries filled with phosphate buffer containing 25 mM SDS and buffered at pH 6.1. Resolution of the diphosphorylated isomers can be significantly improved by using polyacrylamide coated capillaries. In coated capillaries electroosmotic flow is negligible and the migration order of the isomers is reversed. This allows for a longer period of interaction between the diphosphorylated isomers and the micelle and therefore selectivity is improved. Efficiency of all peptide isomers was also improved in coated capillaries due to reduced adsorption to the capillary wall.

Capillary electrophoretic separation of nucleotide isomers via complexation with cyclodextrin and borate.

The electrophoretic behaviour of monophosphorylated nucleotide isomers can be manipulated using complex-forming reactions with beta-cyclodextrin (beta-CD) and borate. Resolution of the 2'- and 3'-isomers of nucleotides is possible when the electrophoresis buffer contains 10 mM CD. The effect of beta-CD concentration on electrophoretic mobility is used to calculate the formation constant, K, of beta-CD-nucleotide complexes. The 3'-isomer of adenosine monophosphate (AMP) forms the strongest complex with beta-CD probably as a result of hydrogen bonding between the phosphate group of AMP and hydroxyls of beta-CD. In addition, complexation of 5'-nucleotides with borate increases the migration time window and leads to better separation. Complex-forming reactions of guanosine monophosphate and uridine monophosphate are shown to be strongly dependent on buffer pH. A mixture of 12 monophosphorylated nucleotides can be separated in less than 15 min using a buffer of 20 mM borate-10 mM beta-CD.

Effect of detergents on the electrophoretic behaviour of plasma apolipoproteins in capillary electrophoresis.

The influence of various detergents on the capillary electrophoretic behaviour of plasma apolipoproteins was studied. Electrophoretic mobility increased in the presence of anionic detergents sodium deoxycholate (DOC) and sodium dodecyl sulfate (SDS), and decreased in the presence of non-ionic Triton X-100. Apolipoproteins from plasma low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) exhibited different affinities for DOC and SDS. Optimal separation and reproducibility of HDL and LDL apolipoproteins was obtained using high-pH buffers containing SDS. Good resolution of very-low-density lipoprotein (VLDL) apolipoproteins was obtained on addition of either SDS or cetyl trimethylammonium bromide to the running buffer. For VLDL apolipoproteins the use of polyacrylamide coated capillaries yielded better results.

Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.