Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method.

A method for the preparation of new aminated agarose gels containing monoaminoethyl-N-aminoethyl structures, MANA-agarose gels, has been developed. These gels contain primary amino groups with a very low pK value (6.8). In addition to that, we have been able to prepare very highly activated gels (e.g., 10% agarose gels containing up to 200 mu Eq of primary amines per milliliter). These two properties make these activated supports suitable for performing novel and interesting methods for protein immobilizations via very mild carbodiimide activation of carboxy groups. For example, very effective coupling reactions can be performed at pH 5.0-6.0 in the presence of low concentrations of activating agent, e.g., 1 mM. By using a model industrial enzyme, beta-galactosidase from Aspergillus oryzae, we have been able to demonstrate the excellent prospects of these novel activated supports.