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.

Microcalorimetric Studies of the Interactions of Lysozyme with Immobilized Metal Ions: Effects of Ion, pH Value, and Salt Concentration

“…While the (c) process involves the formation of a coordination bond between imidazole and the immobilized metal ion and a nonspecific interaction, the nonspecific interaction should not be neglected as the electrostatic repulsion between protein and adsorbents under no salt present. As can be seen from the literature (21,(31)(32)(33), the negative charge of the CS-IDA-Fe(III) is higher than that of CS-IDA-Cu(II). Therefore, the heat required to overcome the nonspecific interaction (electrostatic repulsion) of Fe(III) is higher than of Cu(II) at pH 8.5, where lysozyme is negatively charged.…”

“…The nonspecific interaction can be affected by the charge density and binding capacity of metal ions on the gel. The literature has also reported (20,(31)(32)(33) that the negative charge on the Sepharose gel was Fe(III) higher than Cu(II).…”

“…The electron donor groups of protein include the imidazole side chain of histidine, the sulfhydryl group of cysteine, and residue of phosphorylated serine. The specific interaction of a protein with immobilized metal ions frequently varies with the topography of the protein surface and the chemistry and physical conditions of the interaction (2)(3)(4)(5)(6)(7)(8).…”

Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins

“…Advantage can also be taken of the relative metal ion stability constant (as the average logb value) in selecting such IMAC systems. Both potentiometric titration and isothermal titration microcalorimetry are particularly useful in characterising different types of soft/borderline/hard metal ion systems with immobilised chelating ligands system, and enable the respective hydrolytic complexes that form with different immobilized metal ion chelating complexes (IMCC's) to be properly defined (Zachariou et al, , 1997Hearn, 2000). Indicative of the potential of these approaches have been various investigations with synthetic peptides (Nakagawa et al, 1988;Porath, 1988a,b Kronina et al, 1999, as well as studies with a-interferon -related peptides (Sulkowski 1989), whilst the elegant exploratory studies related to bovine growth hormone (Suh et al, 1991) has been elaborated above.…”

Potentiometric, kinetic, and thermodynamic investigations into Cu2+ ion binding properties of vinyl imidazole containing IMAC adsorbent

“…Only limited studies have concentrated on the acid-base and metal binding properties of new and traditional chelating adsorbents and on changes in the physicochemical properties of immobilized metal chelate complexes. [21][22][23][24] Because of the weak interaction between vinyl imidazole and Cu 21 ions in the solid IMA chromatography matrixes, no data exist on complex stability and complex stoichiometry in literatures. Until now, the strength of the Cu 21 -vinyl imidazole complex has been determined only in the water-soluble linear poly (1-vinyl imidazole) systems.…”