FRACTIONATION OF THE GENETIC-VARIANTS OF HUMAN ALPHA(1)-ACID GLYCOPROTEIN IN THE NATIVE FORM BY CHROMATOGRAPHY ON AN IMMOBILIZED COPPER(II)AFFINITY ADSORBENT - HETEROGENEITY OF THE SEPARATE VARIANTS BY ISOELECTROFOCUSING AND BY CONCANAVALIN-A AFFINITY-CHROMATOGRAPHY

Fractionation of the three main genetic variants (F1, S and A) of huma n alpha1-acid glycoprotein (AAG), in their native (sialylated) form, b y chromatography on immobilized copper(II) affinity adsorbent was inve stigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had bee n previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different sample s on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two p rotein peaks, irrespective of the origin of the native AAG sample used . The unbound peak 1 was found to consist of the F1, the S or both var iants, depending on the phenotype of the AAG sample used in the chroma tography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were fou nd to be the same as those originally yielded by the desialylated prot ein. However, comparison of the interactions of native and desialylate d AAG with immobilized copper(II) ions, using an affinity chromatograp hic method and a non-chromatographic equilibrium binding technique, re spectively, showed that desialylation increased the non-specific inter actions of the protein with immobilized copper(II) ions. The AAG varia nts were not fractionated when affinity chromatography was performed u sing immobilized zinc, nickel or cobalt(II) ions, instead of copper. A fter purification of each variant in the sialylated form (F1, S and A) , their respective heterogeneity was studied by analytical isoelectrof ocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants w as investigated by affinity chromatography on immobilized concanavalin A.