Retention behaviour 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 investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained the 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 (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated alpha 1-acid glycoprotein.
Millot
M C
MC
Laboratoire de Physico-Chimie des Biopolymères, Université Paris XII Val de Marne, Créteil, France.
Hervé
F
F
Sébille
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009961:Orosomucoid / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011145:Polyvinyls / Q000737:chemistry*; D012492:Salts; D012794:Sialic Acids / Q000737:chemistry*; D012822:Silicon Dioxide
D000596:Amino Acids; D007093:Imidazoles; D009961:Orosomucoid; D011145:Polyvinyls; D012492:Salts; D012794:Sialic Acids; C072974:polyvinylimidazole; D012822:Silicon Dioxide; D003300:Copper; D019158:N-Acetylneuraminic Acid