Interplay of pH and Binding of Multivalent Metal Ions: Charge Inversion and Reentrant Condensation in Protein Solutions

Abstract

Tuning of protein surface charge is a fundamental mechanism in biological systems. Protein charge is regulated in a physiological context by pH and interaction with counterions. We report on charge inversion and the related reentrant condensation in solutions of globular proteins with different multivalent metal cations. In particular, we focus on the changes in phase behavior and charge regulation due to pH effects caused by hydrolysis of metal ions. For several proteins and metal salts, charge inversion as measured by electrophoretic light scattering is found to be a universal phenomenon, the extent of which is dependent on the specific protein-salt combination. Reentrant phase diagrams show a much narrower phase-separated regime for acidic salts such as AlCl3 and FeCl3 compared to neutral salts such as YCl3 or LaCl3. The differences between acidic and neutral salts can be explained by the interplay of pH effects and binding of the multivalent counterions. The experimental findings are reproduced with good agreement by an analytical model for protein charging taking into account ion condensation, metal ion hydrolysis, and interaction with charged amino acid side chains on the protein surface. Finally, the relationship of charge inversion and reentrant condensation is discussed, suggesting that pH variation in combination with multivalent cations provides control over both attractive and repulsive interactions between proteins.

Anisotropic protein–protein interactions due to ion binding

“…by continuum electrostatics at the Debye-Hückel level. Multivalent salt, however, leads to qualitatively different behavior and both experiment and simulation with explicit salt particles predict attractive electrostatics even between proteins of high, equal charge [3,32]. Fig.…”

“…Protons, however, may be in competition for binding sites with other ions and solutes in the solution. For example, recent work shows that trivalent cations such as lanthane and yttrium can reverse the charge of human serum albumin by strong binding to carboxyl groups which in turn influence protein-protein interactions and phase equilibria [2][3][4]. Large ions with a low surface charge density such as thiocyanate or tetra-alkylammonium may bind to hydrophobic surface patches, contributing to the effective electrostatic potential emanating from the protein [5][6][7][8].…”

Competing Salt Effects on Phase Behavior of Protein Solutions: Tailoring of Protein Interaction by the Binding of Multivalent Ions and Charge Screening

“…Strong pH shifts are observed in systems where the metal ions undergo hydrolysis (e.g., FeCl 3 , AlCl 3 ). 27 We remark that no dialysis or other purification of the samples was necessary, since the conclusions of the study are based on qualitative trends with increasing salt concentration, and the amount of counterions dissociating from the protein surface is expected to be lower than the concentration of monovalent salt.…”

“…An analytical model for the binding of multivalent ions to a spherical particle, taking into account charge regulation of surface groups, counterion binding as well as pH effects due to hydrolysis of multivalent metal ions, has been developed recently. 27 The scheme is based on association reactions between the present ions and surface sites, the binding to which is further dependent on the total particle charge. A detailed description of the method as well as the choice of suitable model parameters solely based on literature values and theoretical estimates can be found in ref 27. In this coarse-grained model, the addition of monovalent salt varies the surface potential via the Debye screening length κ and thus changes the binding equilibria of multivalent ions.…”

Anomalous Protein–Protein Interactions in Multivalent Salt Solution

“…This anomalous trend, caused by charge reversal, has been shown to be able to induce clustering, liquid-liquid phase separation or crystallization. 12,18,[21][22][23][24][25] The ability to control the phase behavior of protein dispersions show promising opportunities, for example for the production of high quality protein crystals, required for protein structure determination.…”