Hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of pH.

Hydroxyapatite is a form of calcium phosphate that has long been used in the chromatographic separation of proteins andDNA [1]. Hydroxyapatite is best known as a crystalline material but is now available in a range of ceramic derivatives that are vastly superior in terms of flow rate, stability, and reproducibility over many cycles of use. These developments have led to a renewed interest in the use of this media with unique separation properties. This report aims to further extend the usefulness of hydroxyapatite for the purification of proteins. The adsorption of proteins to hydroxyapatite is complicated because it involves both anionic and cationic exchange. The Ca2þ functional groups can interact with carboxylate residues at the protein surface while the PO 4 functional groups can interact with basic protein residues [2]. Proteins are most commonly adsorbed in a low concentration (10–25mM) of phosphate buffer, although some acidic proteins are adsorbed only if loaded in water, saline, or a nonphosphate buffer [3]. Proteins are usually eluted by an increasing phosphate gradient, although gradients of Ca2þ, Mg2þ, or Cl ions are also useful, especially for the selective elution of basic proteins [2]. When using phosphate, acidic proteins are more readily eluted than basic proteins, although the phosphate concentration required to elute any protein can be reduced by raising the pH [4]. A mixture of proteins bound to hydroxyapatite can be fractionated by a series of phosphate wash steps of increasing pH [5]. However, phosphate is a weak buffer outside the 6.0–7.5 pH range, yet strong buffering is desirable when using hydroxyapatite to avoid the local pH changes that occur in the highly polarized environment at the surface [6]. Analysis of the effect of altering the pH upon protein elution would therefore benefit from improved buffering because the buffering capacity of the phosphate rapidly decreases away from neutrality. In the present paper, a simple procedure is described that extends the phosphate-mediated hydroxyapatite elution of proteins over a wider pH range while maintaining a properly buffered environment. Others have not previously reported the approach of using a second buffer system to broaden the pH range of separation with hydroxyapatite media.