20] Protein chromatography on hydroxyapatite columns

Abstract

The use of hydroxyapatite (HA) columns for protein chromatography has been introduced by Tiselius et al. He also proposed a mechanism for protein adsorption to and desorption from HA, since the principles of ion-exchange chromatography are not applicable to HA chromatography. Furthermore, the roles of protein fine structure and of specific polar groups have been probed. This last study included proteins in which individual polar groups were modified specifically, along with those in which very extensive numbers of amino or carboxyl groups are modified with (1) retention of the same charge, (2) inversion of the charge, or (3) annihilation of the charge. As a result of this work, a mechanism of protein-HA interaction has been deduced, which is based on three fundamental conclusions: 1. Adsorption and elution cannot be regarded as simple reversals of a single process. 2. Amino and carboxyl groups act differently in the adsorption of proteins to HA. 3. Elutions of basic and acidic proteins by different salts follow different mechanisms.