High-performance adsorption chromatography of proteins on deformed non-porous agarose beads coated with insoluble metal compounds : I. Coating: Ferric oxyhydroxide with stoichiometrically bound phosphate

Abstract

A new support for adsorption chromatography of proteins based on crosslinked agarose gels containing ferric (hydr)oxide is described. Two methods have been used for immobilization of the ferric (hydr)oxide on agarose beads; namely,entrapment of preformed ferric oxide particles in connection with the preparation of macroporous agarose beads and precipitation of ferric oxyhydroxide on and in preformed non-porous agarose beads. In the former method many of the adsorption sites of the ferric oxide may become sterically blocked, resulting in chromatographic properties which are similar to those of cation exchangers. Therefore, attention was focused on the second method. Upon using compressed beds of non-porous agarose beads the resolution became independent of the flow-rate, even when the bead diameter was relatively large. When ferric oxyhydroxide agarose is equilibrated with phosphate buffer, some phosphate becomes stoichiometrically and irreversibly bound to the matrix. The separation mechanism of this ferric oxyhydroxide phosphate agarose is discussed. It differs from those of ion-exchange, hydrophobic-interaction, and molecular-sieve chromatography. One characteristic is that phosphate buffers are the most efficient eluting agents. Chromatography on ferric oxyhydroxide phosphate-agarose is accordingly a good complement to these three conventional methods. Some chromatographic properties of the adsorbents are described (resolution as a function of gradient time, flow-rate and protein load; and the pH dependence of the adsorption). A successful separation of a model mixture of six proteins was achieved. Purification of a commercial preparation of β-glucosidase is also presented.