Interaction mechanisms between insulin and N-acetylneuraminic acid in affinity chromatography.

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylaminoethyl (DEAE) groups in order to neutralize negatively charged silanol groups at the silica surface and in this way to minimize non-specific interactions between silica and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports [silica-dextran-DEAE (SID)] are easily functionalized by grafting N-acetylneuramic acid (NANA), extracted from edible birds' nests, using conventional coupling methods. The performance of supports bearing NANA was studied by high-performance liquid affinity chromatography of insulin, the hypoglycaemic peptide hormone of the human organism. The study showed that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. The influence of different physico-chemical parameters (pH, temperature and insulin concentration) on insulin retention on the support was studied. This allowed the optimization of the conditions of adsorption and a better understanding of the interaction mechanisms between insulin and NANA as a biospecific ligand.