2-STEP CHROMATOGRAPHIC PURIFICATION OF HUMAN PLASMA ALPHA(1)-ACID GLYCOPROTEIN - ITS APPLICATION TO THE PURIFICATION OF RARE PHENOTYPE SAMPLES OF THE PROTEIN AND THEIR STUDY BY CHROMATOGRAPHY ON IMMOBILIZED METAL CHELATE AFFINITY ADSORBENT

alpha(1)-Acid glycoprotein (AAG) or orosomucoid was purified to homoge neity from human plasma by a separate two-step method using chromatogr aphy on immobilized Cibacron Blue F3G-A to cross-linked agarose and ch romatography on hydroxyapatite. The conditions for the pre-purificatio n of AAG by chromatography on immobilized Cibacron Blue F3G-A were fir st optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 4 0 mi sample of human plasma. This method was applied to the purificati on of AAG samples corresponding to the three main phenotypes of the pr otein (F1S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampho lytes showed that the microheterogeneity of the purified F1S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding pl asma, thus suggesting that no apparent desialylation of the glycoprote in occurred during the purification steps. This method was also applie d to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/AAD, S/A*X(o) and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded b y the second gene of AAG (i.e. the A, AD, X(o) and C1 variants) strong ly bound to immobilized Cu(LI) ions. These results suggested that chro matography on an immobilized affinity Cu(II) adsorbent could be helpfu l to distinguish between the respective products of the two highly pol ymorphic genes which code for human AAG.