18] Chromatography of proteins on hydroxyapatite

Abstract

Publisher Summary This chapter discusses the mechanism of adsorption of proteins on hydroxyapatite (HA), particularly as far as the interaction of amino acid side groups with adsorption sites on HA crystals is concerned. Two different types of adsorbing sites exist on the surface of HA crystals: calcium sites and phosphate sites. The former appear to bind acidic groups, carboxyls, and phosphates; the latter bind basic groups. This picture fits with the known amphotheric character of HA crystals. Elution is caused by anions, which compete with the carboxyl or phosphate groups of proteins for the calcium sites of HA, or by cations, which compete with the basic groups of proteins for the phosphate groups of HA. Investigations have shown that: (1) the elution of proteins from HA columns can be obtained using a number of eluents other than the usual phosphate buffer, pH 6.8; this leads to a remarkable increase in the potentialities of the method and (2) the chromatographic behavior of basic, neutral, and acidic proteins on HA columns operated with different elution systems can be predicted to a considerable extent, thus permitting a less empirical approach to separation problems; in turn, the elution molarities of proteins in different solvent systems can be used to identify the nature of the interacting amino acid side groups.