Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter.

The human red cell glucose transporter (Glut1) is a transmembrane protein. Monomeric Glut1 was purified by ion-exchange chromatography in the presence of the non-ionic detergent n-dodecyl octaoxyethylene (C12E8). For focusing, the ionic strength of the solution of C12E8-Glut1 complexes with co-purified lipids was lowered by dialysis, the detergent concentration was increased and carrier ampholytes were added. Focusing was done for 5 min at 3000 V in a methyl cellulose-coated glass capillary (50 microns I.D.). The anolyte H3PO4 was then replaced by NaOH for mobilization towards the anode. Absorbance monitoring at 280 nm showed two groups of zones at pH 6 and 8. Similarly, isoelectric focusing in a rotating quartz tube (3 mm I.D.) gave Glut1 zones at pH 5.5 and 8.0. Phosphorus analysis revealed that the Glut1 zone at pH 8 contained more phospholipids than did the other one. The above results together with previously determined and calculated isoelectric points (pI) of Glut1 indicate that the Glut1 at pH 8 is monomeric and that the zone at pH 5.5-6 represents oligomeric materials. The pI 8.0 at 22 degrees C applies for monomeric Glut1 in the absence of urea. The results exemplify that capillary isoelectric focusing of hydrophobic membrane proteins is possible.