Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Abstract

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.

Figure 1

Representative electropherogram (A) and c(s) sedimentation coefficient distribution (B) of HEWL in 10 mM acetate, 50 mM NaCl at pH 5.0. The electrophoretic mobility was calculated using the elution times of the EOF marker, (t_EOF) and HEWL (t_HEWL), using (2). The c(s) distribution of HEWL shows a single peak at 1.9 s with no evidence of aggregation at higher s values. The diffusion coefficient was calculated using the sedimentation coefficient (3).

Figure 2

The effective charge (Q*) of hen-egg white lysozyme (HEWL) in different Hofmeister salt solutions. Panel A: The HEWL Q* in NaCl (), NaNO₃ (), NaClO₄ (), Na₂SO₄ (), CsCl (), GdnHCl (), and CaCl₂ () as a function of ion normality. Note that the NaCl, CsCl, GdnHCl, and CaCl₂ data coincide. The lines through data series are presented as guides to the eye only. Panel B: At 50 mM ion normality, Q* is dependent on anion but not cation, identity. Panel C: Plotting HEWL Q* in NaCl () and CaCl₂ () versus ionic strength (I) results in a higher Q* in CaCl₂, suggestive of calcium binding to HEWL; however, the trends coincide when plotted versus ion normality (Panel A).

Figure 3

Two views of the HEWL electrostatic surface, rotated 180° along the horizontal axis. The surface is heterogeneous with respect to charge. The largest charged region (negative, red) appears as part of the cleft in the upper structure. Positively charged regions are shown in blue.