Small unilamellar vesicles containing glycophorin A. Chemical characterization and proton nuclear magnetic resonance studies

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Abstract
Glycophorin A, a major glycoprotein of the human red blood cell, is reconstituted in small lipid vesicles (250-300 .ANG. in diameter) by using cholate detergent solubilization followed by rapid removal of cholate on a molecular sieve column. The extent of glycophorin incorporation is critically dependent on the amount of cholate used, with higher amounts yielding vesicles with higher percentages of glycophorin. Vesicles with as much as 1 molecule of protein/20 molecules of lipid can be prepared. Data on the vesicles obtained by using hydrolytic enzymes such as neuraminidase and trypsin, combined with amino acid analysis, suggest that glycophorin is incorporated in a transbilayer fashion with a high fraction of the molecules oriented with the carbohydrate-containing amino terminus to the vesicle exterior. Interaction of the protein with the hydrophobic portion of the bilayer is apparent in 1H NMR spectra, and lipid line-width increases were used to characterize the strength and stoichiometry of interaction. Glycophorin affects directly as many as 40 lipid molecules/molecule of protein; the magnitude of the effects is not large.