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Hydroxyapatite

Hydroxyapatite is a special crystalline form of calcium phosphate that has been used in the separation of biological molecules. The adsorbent consists of a mosaic of crystals that result from a combination of phosphate and calcium salts after a number of steps of wet alkaline treatment and heat aging. The application of hydroxyapatite to the field of protein separation was first introduced by Tiselius et al. (18). The standard procedures for the operation of hydroxyapatite columns were developed by Bernardi and Gorbunoff (19,20) using a number of different proteins in several solvent systems, Bernardi also proposed a mechanism for protein adsorption to, and desorption from, hydroxyapatite.

Proteins bind reversibly to hydroxyapatite by a complex ionic mechanism different from the principle of ionexchange chromatography (19,20). Both the amino and carboxyl groups on the protein molecules act in the adsorption mechanism. The adsorption of proteins to hy-droxyapatite via the amino groups is the result of their positive charges and the negative charge on the adsorbent when the column is equilibrated in phosphate buffer. The surface of hydroxyapatite crystals consists of sites with both positive (calcium) and negative (phosphate) charges. When the hydroxyapatite columns are equilibrated in phosphate buffers, normally at pH 6.8, the surface of the adsorbent can be regarded as negative because of partial neutralization of the positive calcium loci by phosphate ions. The carboxyl groups act in two ways. First, they are repelled electrostatically from the negative charge of the adsorbent; second, they bind specifically by complex formation with the calcium sites.

The elution of basic proteins from hydroxyapatite is generally carried out by specific displacement with Ca2 + and Mg2 + ions that neutralize the negative charges of the hydroxyapatite phosphate sites by complex formation. Acidic proteins are eluted by displacement of their car-boxyl groups from hydroxyapatite calcium sites by ions, such as fluoride or phosphate, which form stronger complexes.

The difficulty with using hydroxyapatite in medium-and large-scale purification of biological molecules is the physical nature of the material. The crystals are highly fragile, generating small particles resulting in column clogging and precluding any long-term usage of the column. An improved form of hydroxyapatite crystals has recently been prepared and is commercially available from a number of sources, including Bio-Rad. The new ceramic material from Bio-Rad is spherical and has been reported to be stable both chemically and physically.

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