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Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris-HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.

SummaryThe separation of gJycoproteins as gJycoforms with specific degrees of gJycosyJation has been a problem until now. A technique involving boronate affinity chromatography has been developed to separate a heterogeneous sample of neoglycoprotein, chymotrypsin modified with maltose via reductive amination, into individual fractions with different degrees of glycosylation. Low-molecular-mass polyhydroxyl compounds, such as tris(hydroxymethyl)aminomethane (Tris), pentaerythritol, and triethanolamine proved efficient eluents clue to their ability to form strong tridentate complexes with the boronate ligand. Compounds leading to either: too strong interaction with the boronate hgand (e. g. D-sorbitol and polyvinly alcohol), or too weak or no interaction (e.g. dextran) were not suitable eluents. The study provided the opportunity to probe further into the effect of glycosylation on the function of glycoproteins.