Affinity-repulsion chromatography. Principle and application to lectins.

The interactions of proteins with their immobilized ligands in an electrically charged microenvironment were studied. The binding of lectins to erythrocytes and to affinity matrices was used as a model system. Lectins bind and agglutinate erythrocytes in the presence of at least 10 mM NaCl or 1 mM CaCl2, but not in deionized water. The salt dependence of the agglutination process is due to the ability of salts to provide counterions neutralizing the forces of repulsion between the electrostatic charges of similar sign present on the erythrocyte cell surface and on the lectins. The same salt dependence is observed for the binding of lectins to affinity matrices. These observations are the basis of a protein separation process coined affinity-repulsion chromatography in which the electrostatic charges present, or purposely introduced, on affinity matrices are exploited and allow the elution, by electrostatic repulsion, of proteins carrying electrostatic charges of the same sign as that of the matrix. In this process, proteins are loaded on the affinity matrix in a salt solution and eluted with deionized water. Affinity-repulsion chromatography has been successfully applied here to the isolation of several lectins. Its physicochemical basis, merits, and potential applications are discussed.