Molecular recognition in hydroxyapatite chromatography and ion exchange chromatography of proteins

Molecular recognition in hydroxyapatite chromatography (HAC) and ion exchange chromatography (IEC) is investigated. A fast and simple method for obtaining important information on the number of binding sites from linear gradient elution experiments (salt concentration is increased linearly at a fixed mobile phase pH) is first described. Linear gradient elution experiments for HAC and IEC were carried out with beta -lactoglobulin (Lg) and Ribonuclease A (RNaseA) as model proteins. The experimental data were analyzed on the basis of the above-mentioned method. The peak salt concentration I-R and the number of binding sites B in IEC decrease as the mobile phase pH approaches the isoelectric points pi (Lg= 5.1-5.3, RNaseA =9.7) for both Lg and RNaseA. The I-R and B values of Lg in HAC decrease as the mobile phase pH increases. Although the I-R values of RNaseA in HAC decrease with an increase in the mobile phase pH, the B values are constant (B ca. 5) and did not depend on the mobile phase pH. Two genetic variant forms of Lg, beta -lactoglobulin A and beta -lactoglobulin B, are not separated on HAC and cation-exchange chromatography. The two proteins are separated only on anion-exchange chromatography. On the basis of these experimental findings the molecular recognition mechanism of HAC with Lg and RNaseA is discussed in comparison with the mechanism in IEC.