Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated sepharose: study of the active groups involved in nonspecific adsorption.
Fibrinogen binds specifically to Lens culinaris agglutinin coupled to CNBr-activated Sepharose. However, a fraction of the retained fibrinogen remains tightly bound to the gel and is eluted only by electrophoretic desorption. The irreversible binding of fibrinogen results from the interaction of fibrinogen specifically bound to the immobilized lectin with some reactive groups still present on the Sepharose matrix. Therefore, the active groups present on lectin-Sepharose after different treatments and their influence on the irreversible binding of fibrinogen have been studied. After the coupling step some cyanate esters remain on the gel, but they are neutralized under all the conditions studied. In addition, imidocarbonates formed under the basic conditions used to activate Sepharose, and carbonates resulting from acid treatment of the gel, are also present. Carbonates seem to be the main active groups involved in the irreversible binding of fibrinogen to lectin-Sepharose. Imidocarbonates also contribute to the nonspecific binding, although to a lesser extent than carbonates. Treatment of CNBr-activated Sepharose with 0.1 M HCl prior to the coupling step and neutralization, after coupling, with 0.1 M ethanolamine, pH 9.5, for 24 h at room temperature reduce the nonspecific binding to less than 9% of the fibrinogen fraction retained by the column. This percentage is appreciably smaller than that obtained by neutralization for 2 h at room temperature with either 0.1 M Tris-HCl, pH 8.0 (congruent to 66%), or 1 M ethanolamine, pH 9.0 (congruent to 23%).
Tercero
J C
JC
Instituto de Química-Física Rocasolano, Madrid, Spain.
Diaz-Mauriño
T
T
D000327:Adsorption; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D003488:Cyanogen Bromide; D005340:Fibrinogen / Q000302:isolation & purification*; D037102:Lectins; D037121:Plant Lectins; D012685:Sepharose
D037102:Lectins; D037121:Plant Lectins; C033619:lentil lectin; D005340:Fibrinogen; D012685:Sepharose; D003488:Cyanogen Bromide