29] Chromatography of proteins on hydroxyapatite

Abstract

Publisher Summary This chapter discusses several factors that need to be considered for the chromatography of proteins on hydroxyapatite (HA) columns. To understand the mechanism of adsorption and elution of proteins, it is important to know which chemical groups are involved in the protein–HA interaction. In proteins, the negative groups, carboxyl and phosphate, and positive groups can interact with HA. The binding sites for the negatively charged groups of proteins are, in all likelihood, the calcium ions at the surface of the HA crystals. Dyes forming sparingly soluble salts with calcium ions, such as the crystal violet and alizarin dyes, have a strong affinity for HA. In contrast with the negative groups, binding sites for the positive groups of proteins are not known. These may be the same calcium ions or, more likely, negatively charged groups. It is important to stress that HA crystals are amphoteric and that isoelectric points of different HA preparations are found to range from 6.5 to 10.2.