Affinity-based reversed micellar extraction and separation (ARMES) has
proven effective in separating glycoproteins from nonglycosylated pro
teins from natural sources. The ability of ARMES to resolve closely re
lated glycoproteins is of paramount importance if ARMES is to be used
in glycoform resolution. It is demonstrated that ARMES can resolve the
structurally similar soybean peroxidase (SBP; MW 37 kDa, pI 4.1) and
alpha(1)-acid glycoprotein (AGP; MW 43 kDa, pI 3.7), both of which hav
e affinity for Concanavalin A (Con A) (the affinity ligand). SBP was a
lmost exclusively extracted at pH 8 and above, with a separation facto
r greater than 50 (resolution similar to 20), far better than was poss
ible using Con A affinity chromatography (R similar to 0.25, separatio
n factor similar to 2). Model calculations suggest that differences in
affinity measured by an equilibrium-building assay cannot account for
the favorable extraction of SBP over AGP at higher pH. Hydrophobic in
teractions and/or charge shielding appear to affect partitioning of th
e lectin-glycoprotein complexes and add greatly to the selectivity of
extraction in ARMES, especially at higher pH values.