Potentiometric Investigations into the Acid−Base and Metal Ion Binding Properties of Immobilized Metal Ion Affinity Chromatographic (IMAC) Adsorbents

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.

Phosphoserine and specific types of its coordination in copper(II) and adenosine nucleotides systems – Potentiometric and spectroscopic studies

“…For phosphoserine the values of successive protonation constants: log K 1 = 10.03, log K 2 = 6.04, log K 3 = 2.63 and log K 4 = 2.12, Table 1, determined from the computer analysis of the titration data correspond to the protonation of the subsequent groups: -NH 2 , -PO 2À 4 , -COO À and the second site at the phosphate group, respectively [25,26]. The sequence of the AP nucleotides protonation was: log K 1 of about 6.5 -the protonation of phosphate group, log K 2 of about 4.0 -the protonation of N(1) [27][28][29][30][31], Table 1, and log K 3 0.4 for H 3 AMP + , 1.02 for H 3 ADP and 1.7 for H 3 ATP À -second site at the phosphate group in each AP, respectively [32].…”

Protein Interaction with Immobilized Metal Ion Affinity Ligands under High Ionic Strength Conditions

“…Consequently, a of HEWL to Cu 2/ -IDA adsorbent takes a finite time to reach equilibrium, typically less than 30 min with number of alternative isotherm equations have been applied to protein adsorption. In particular, the Freun-the batch systems employed, and that the time taken to reach equilibrium was not strongly influenced by the dlich model (22) relates the adsorbed concentration as an exponential function of the solution concentration salt concentration of the buffer, although the maximum value of the q* versus t relationship was affected by the by the two-fitted parameters K k and n, i.e., neity of the adsorbent surface, (iii) the compositional or conformational heterogeneity of the protein surface, (iv) the propensity of proteins to associate into macromolecular aggregates, and (v) the participation of secondary protein-solvent or protein-ion equilibria as well as ligand-solvent or ligand-ion equilibria which lead to the binding of the protein to the ligand in a range of different energy states (4,26). As a consequence, when the adsorption behavior for protein-ion exchange and protein-affinity adsorbent interactions has been evaluated in terms of the singlecomponent Langmuir model, investigators (17)(18)(19)(26)(27)(28) tion, in particular, permits a more detailed evaluation KH 2 PO 4 buffer, pH 7.0, containing 0.2 M NaCl (l) and 2 M NaCl (᭺).…”

Theoretical pKa prediction of O-phosphoserine in aqueous solution

“…The only reliable guess, obtained from vibrational spectra in the range of the phosphate group fundamentals, places it much below 1.7 [22]. The three consecutive dissociation steps found in potentiometric experiments correspond to the COOH, HPO À 4 , and NH þ 3 dissociation [2,21,22]. Following our recent study of the dissociation constants of phosphoric(V) acid [13], in this Letter we present the results of a Density Functional Theory (DFT) determination of P-Ser dissociation constants by the use of PCM calculations for solvation free energies and show that inclusion of discrete water molecules is necessary to stabilize the intermediate P-Ser forms against intramolecular proton transfer from NH þ 3 to the phosphate group that commonly occurs in the gas phase optimizations [23].…”

“…Also, potentiometric determination of accurate pK a values below ca. 1.5 is difficult [2]. Another commonly encountered problem is the ascription of microscopic ionization constants to the measured macroscopic pK a s [1,3].…”

“…A similar case of an a-amino acid with an ionisable side group, cysteine, was already investigated with ab initio methods [3]. The case of P-Ser, even though the number of species in the dissociation pathway is larger, seems to be less problematic, as the consecutive pK a s are well separated and the exact sequence of dissociation processes is well known [2,[20][21][22]. The five principal ionic species in aqueous solution, as well as the neutral form, are shown in Scheme 1.…”