Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin.

High-performance immobilized-metal affinity and hydroxyapatite chromatography were employed to investigate the engineered subtilisin S1 binding site microenvironment. Although these methods are classified as affinity techniques, unlike traditional affinity columns, both are capable of probing the entire surface of a molecule. The metal chelate study employed gradient elution to assemble retention maps for a wide range of mobile-phase pH. Resolution of single substitution variants was achieved at the optimum mobile-phase pH. A total of four metals were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. Hydroxyapatite chromatography, albeit an established technique, has only recently been developed as a high-performance chromatographic method. Gradient elution separations were performed to determine selectivity. Immobilized-metal affinity chromatography was found to be the more effective method for the separation of site-specific variants.