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

✍ Scribed by Ronald L. Schnaar; T.Flint Sparks; Saul Roseman

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 910 KB
• Volume: 79
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90425-0

✦ Synopsis

~finity chromatogmphy ligands cont~ning 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 in~o~o~tion varied with the ligand used in the coupting reaction. The pH and the concent~tion 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.

The cyanogen bromide procedure is widely used for the preparation of insolubilized molecules as affinity chromatographic adsorbents or as biological probes. Insoluble polysaccha~des are treated with cyanogen bromide to "activate" the glycans, followed by coupling of the activated polymers with suitable ligands containing amino groups (l-3). While the ligand-polysaccharide bonds formed in these reactions have been partially characterized, quantitation of the different chemical species and identification of by-products has not been reported (4-6). Several recent reports note that the ligand-polysaccharide products contain cationic charge (7-1 l).In fact, Nishikawa and Bailon (l&11) have indicated that this cationic charge may lead to spurious results in the sense that polymers will adsorb anions such as proteins in a nonspecific manner, independent of the ligand being used for affinity chromatography.