RETENTION BEHAVIOR OF PROTEINS ON POLY(VINYLIMIDAZOLE)-COPPER(II) COMPLEXES SUPPORTED ON SILICA - APPLICATION TO THE FRACTIONATION OF DESIALYLATED HUMAN ALPHA(1)-ACID GLYCOPROTEIN VARIANTS

The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was inves tigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary str ucture were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results o btained with proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essential ly ionic and/or hydrophobic) interactions took place between the prote ins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary p hase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the prot eins and the supports via donor-acceptor interactions was less favoura ble, proteins were eluted from the columns without the addition of a c ompeting agent in the mobile phase. With respect to the binding of the se proteins, ionic and/or hydrophobic interactions were no longer negl igible during the chromatographic process and,the retention of the mac romolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractiona tion of the three main genetic variants of desialylated alpha(1)-acid glycoprotein.