A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm

Abstract

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 [mu]mol protein/ml of gel are obtained by reacting protein (0.5-5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH <= 7.4, <= 25 [deg]C). Protein is released from the gel, however, by treatment at 25 [deg]C with solutions containing nucleophiles such as 1 imidazole-glycine, pH 7.4, for 4 h, or 1 hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.