Cyanogen bromide activation of polysaccharides. Effects of reaction conditions on cationic charge and ligand content

Affinity chromatography ligands containing amino groups are commonly linked to cyanogen bromide-activated insoluble polysaccharides. This method introduces cationic charge into the insoluble products which may be sufficient to interfere with the use of these materials for affinity chromatography. The present studies were designed to measure and minimize this potential artifact. The cationic charge of the products was determined simply and rapidly by measuring the quantity of labeled orthophosphate bound by the beads. The pH used during activation was critical in determining the extent of ligand incorporation and the cationic charge of the product; high pH gave increasing cationic charge. However, the extent of ligand or charge incorporation varied with the ligand used in the coupling reaction. The pH and the concentration of the ligand in the coupling step also had marked but different effects on the results, depending on the ligand used. It was possible to select conditions for maximizing incorporation while minimizing cationic charge of the products for a given ligand.