Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose. Effect of protein charge on immobilization

• Published in: Biochimica et biophysica acta Vol. 670; no. 2; p. 163
• Main Authors: Frost, R G, Monthony, J F, Engelhorn, S C, Siebert, C J
• Format: Journal Article
• Language: English
• Published: Netherlands 29-09-1981
• Subjects: Animals; Cattle; Chemical Phenomena; Chemistry; Chromatography, Affinity - methods; Concanavalin A; gamma-Globulins; Hemoglobins; Humans; Hydrogen-Ion Concentration; Isoelectric Point; Ovalbumin; Polysaccharides; Proteins; Sepharose - analogs & derivatives; Serum Albumin, Bovine; Transferrin
• ISSN: 0006-3002
• DOI: 10.1016/0005-2795(81)90004-0

An uncharged N-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 N-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.