No abstract

The aim of these studies was to investigate the use of an optimized potentiometric system for the determination of the acid−base dissociation constants of immobilized chelating ligands and the stability constants of the derived metal ion complexes used in the immobilized metal ion affinity chromatographic (IMAC) analysis and purification of peptides and proteins. In particular, potentiometric comparison of the immobilized iminodiacetic acid (IDA) ligand system with a range of hard metal ions, such as Fe3+, Al3+, Yb3+ or Ca2+, and the borderline metal ion Cu2+, has been determined with the commonly used chromatographic support material, Sepharose CL-4B. The values of these derived constants have been compared to the values of the corresponding constants for the free chelating ligand and their metal ion complexes in solution. In addition, the same potentiometric methods have been employed for the determination of the acid−base dissociation constants and stability constants of the derived metal ion complexes for the tridentate ligand, O−phosphoserine (OPS). Besides the participation of the metal ion−ligand complexes of the type ML and ML2, the results indicate the participation of hydrolytic complexes of the type M(OH) m L n with some of these IMAC systems. The availability of this information on the physicochemical characteristics of the free and immobilized metal ion chelate complexes should facilitate the interpretation of the binding behavior with peptides and proteins observed with IMAC adsorbents under a variety of adsorption and elution conditions.

The acid-base protonation constants of two recently introduced chelating ligands for protein purification, O-phosphoserine and 8-hydroxyquinoline immobilized onto Sepharose CL-4B, and the stability constants of their derived immobilized metal ion chelate complexes have been determined by potentiometric methods. The data confirm that immobilization thermodynamically constrains the ligands, with the electron-withdrawing characteristics of the group linking the ligand to the support material affecting the magnitude of the stability constant of the immobilized metal ion complex vis-à-vis the free ligand-metal ion complex in solution. The influence of buffer composition, ionic strength, and pH on the stability constant of the immobilized hard metal ion chelate complexes has also been examined. Collectively, the results have confirmed that coordination complexes with stoichiometries other than the simply 1:1 ML-type exist with these systems, with hard metal ions exhibiting a preference for hydrolytic M(OH)(m)L(n) complexes where m or n > 1. These findings on the participation of coordination complexes of different stoichiometry depending on the characteristics of the chelating ligand and metal ion have fundamental implications for the interpretation of immobilized metal ion affinity chromatographic separation of proteins.