Resolution of Glycoproteins by Affinity-Based Reversed Micellar Extraction and Separation

( ) Affinity-based re®ersed micellar extraction and separation ARMES has pro®en effecti®e in separating glycoproteins from nonglycosylated proteins from natural sources. The ability of ARMES to resol®e closely related glycoproteins is of paramount importance if ARMES is to be used in glycoform resolution. It is demonstrated that ARMES ( ) can resol®e the structurally similar soybean peroxidase SBP; MW 37 kDa, pI 4.1 and ( ) a -acid glycoprotein AGP; MW 43 kDa, pI 3.7 , both of which ha®e affinity for Con1 ( ) ( ) cana®alin A Con A the affinity ligand . SBP was almost exclusi®ely extracted at pH 8 ( ) and abo®e, with a separation factor greater than 50 resolution ; 20 , far better than ( ) was possible using Con A affinity chromatography R;0.25, separation factor ; 2 . Model calculations suggest that differences in affinity measured by an equilibrium-building assay cannot account for the fa®orable extraction of SBP o®er AGP at higher pH. Hydrophobic interactions andror charge shielding appear to affect partitioning of the lectin] glycoprotein complexes and add greatly to the selecti®ity of extraction in ARMES, especially at higher pH ®alues.