Characterization by Potentiometric Procedures of the Acid−Base and Metal Binding Properties of Two New Classes of Immobilized Metal Ion Affinity Adsorbents Developed for Protein Purification

Abstract

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.