CHARACTERIZATION BY POTENTIOMETRIC PROCEDURES OF THE ACID-BASE AND METAL-BINDING PROPERTIES OF 2 NEW CLASSES OF IMMOBILIZED METAL-ION AFFINITY ADSORBENTS DEVELOPED FOR PROTEIN-PURIFICATION

The acid-base protonation constants of two recently introduced chelati ng ligands for protein purification, O-phosphoserine and 8-hydroxyquin oline immobilized onto Sepharose CL-4B, and the stability constants of their derived immobilized metal ion chelate complexes have been deter mined by potentiometric methods. The data confirm that immobilization thermodynamically constrains the ligands, with the electron withdrawin g characteristics of the group linking the ligand to the support mater ial affecting the magnitude of the stability constant of the immobiliz ed metal ion complex vis-a-vis the free ligand-metal ion complex in so lution. The influence of buffer composition, ionic strength, and pH on the stability constant of the immobilized hard metal ion chelate comp lexes has also been examined. Collectively, the results have confirmed that coordination complexes with stoichiometries other than the simpl y 1:1 ML-type exist with these systems, with hard metal ions exhibitin g a preference for hydrolytic M(OH)(m)L(n) complexes where m or n > 1. These findings on the participation of coordination complexes of diff erent stoichiometry depending on the characteristics of the chelating ligand and metal ion have fundamental implications for the interpretat ion of immobilized metal ion affinity chromatographic separation of pr oteins.