Shielding of protein-boronate interactions during boronate chromatography of neoglycoproteins.

A method for separating glycoproteins on a boronate column under conditions which suppress the interactions between the protein moiety and the boronic acid ligand has been developed. A model system consisting of non-glycosylated chymotrypsin and maltose-modified chymotrypsin (cht-mal) was utilised in the investigations. Chymotrypsin was chosen as the model protein because of its known interaction with boronate. By coupling maltose to chymotrypsin, a neoglycoprotein was created which has the property of binding to the affinity matrix both via the protein moiety and via the carbohydrate residues. The introduction of a so-called shielding reagent into the buffer solutions during chromatography resulted in the prevention of the protein-boronate interactions while the carbohydrate-boronate interaction was little influenced. Different types of, mainly low-molecular-mass, polyhydroxyl chemicals were screened in order to correlate the shielding efficiency to the chemical structure of the investigated compounds. Polyhydroxyl chemicals with a conformation that allows the formation of tridentate complexes with the boronate anion provided the highest shielding efficiencies.