POTENTIOMETRIC INVESTIGATIONS INTO THE ACID-BASE AND METAL-ION BINDING-PROPERTIES OF IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHIC (IMAC) ADSORBENTS

The aim of these studies was to investigate the use of an optimized po tentiometric system for the determination of the acid-base dissociatio n constants of immobilized chelating ligands and the stability constan ts of the derived metal ion complexes used in the immobilized metal io n affinity chromatographic (IMAC) analysis and purification of peptide s and proteins. In particular, potentiometric comparison of the immobi lized iminodiacetic acid (IDA) ligand system with a range of hard meta l ions, such as Fe3+, Al3+, Yb3+ or Ca2+, and the borderline metal ion Cu2+, has been determined with the commonly used chromatographic supp ort material, Sepharose CL-4B. The values of these derived constants h ave been compared to the values of the corresponding constants for the free chelating ligand and their metal ion complexes in solution. In a ddition, the same potentiometric methods have been employed for the de termination of the acid-base dissociation constants and stability cons tants 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 ML(2), the results indicate the particip ation of hydrolytic complexes of the type M(OH)(m)L(n) with some of th ese IMAC systems. The availability of this information on the physicoc hemical 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 vari ety of adsorption and elution conditions.