Comparison of Methods for Activating Hydroxyl Groups for Preparing Immobilized Biomolecules

Hydroxyl groups of carboxyl-containing matrices, especially cellulose and agarose, can be activated with a wide variety of compounds to make a reactive surface capable of covalently bonding nucleophilic materials. Such materials are widely used in forming affinity chromatography matrices, immobilized enzymes and immunoassays. Our work has been focused on a study of seven major activation reagents: halomethylpyridinium salts, sulfonyl chlorides, cyanogen bromide, carbonyl-diimidazole and other phosgene derived reagents, periodate oxidation/reduction, diazonium salts and activated heterocyclic rings, e.g. epoxides and aziridines. These have been examined for bond stability, active group capacity, stability of attached enzymes, rate of reactions, and ease of handling. No one reagent is suitable for all applications and our study will provide guidelines for the choice of the activating reagent for a wide variety of users.