Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.
Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.
D000409:Alanine / Q000031:analogs & derivatives; D002462:Cell Membrane / Q000032:analysis; D002847:Chromatography, Agarose / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D004912:Erythrocytes / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives; D012967:Sodium Dodecyl Sulfate
D006023:Glycoproteins; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; C011703:N-(3-carboxypropionyl)aminodecyl-sepharose; D012685:Sepharose; D000409:Alanine