Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose

An uncharged $\text{N-}\text{hydroxysuccinimide ester}$ derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5–11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3–5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl₂, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new $\text{N-}\text{hydroxysuccinimide ester}$ derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9–4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.