High-performance hydroxyapatite chromatography of integral membrane proteins and water-soluble proteins in complex with sodium dodecyl sulphate.
Integral membrane proteins from human erythrocytes were fractionated in the presence of sodium dodecyl sulphate (SDS) on four types of high-performance hydroxyapatite columns. A column of 2-microns sintered hydroxyapatite beads from Asahi Optical (Tokyo, Japan) gave the best resolution. With this column, glycophorin was eluted early in a gradient of increasing sodium phosphate buffer concentration, the glucose transporter was eluted later in two zones, one of which contained this protein alone, and the anion transporter was eluted last. Water-soluble proteins applied in complex with SDS also separated reasonably well upon elution. The water-soluble proteins and the membrane proteins were all eluted mainly in the order of increasing polypeptide length, but with considerable individual variation. SDS-polypeptide complexes are probably adsorbed onto hydroxyapatite by the interaction of positively charged amino acid side groups with phosphate ions (at P-sites) and of negatively charged amino acid side groups and polypeptide-bound dodecyl sulphate anions with calcium ions (at C-sites). As a rule, the number of charged side groups and dodecyl sulphate anions, and thus the number of binding sites, increases with the polypeptide chain length, which explains the general order of release of the polypeptides.
Lundahl
P
P
Department of Biochemistry, Biomedical Centre, Uppsala University, Sweden.
Watanabe
Y
Y
Takagi
T
T
D000596:Amino Acids / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D017886:Durapatite; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry; D006801:Humans; D006882:Hydroxyapatites; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012995:Solubility; D014867:Water / Q000737:chemistry
D000596:Amino Acids; D006882:Hydroxyapatites; D008565:Membrane Proteins; D011506:Proteins; D014867:Water; D012967:Sodium Dodecyl Sulfate; D017886:Durapatite