Solubility of lysozyme in polyethylene glycol-electrolyte mixtures: the depletion interaction and ion-specific effects

Abstract

The solubility of aqueous solutions of lysozyme in the presence of polyethylene glycol and various alkaline salts was studied experimentally. The protein-electrolyte mixture was titrated with polyethylene glycol, and when precipitation of the protein occurred, a strong increase of the absorbance at 340 nm was observed. The solubility data were obtained as a function of experimental variables such as protein and electrolyte concentrations, electrolyte type, degree of polymerization of polyethylene glycol, and pH of the solution; the last defines the net charge of the lysozyme. The results indicate that the solubility of lysozyme decreases with the addition of polyethylene glycol; the solubility is lower for a polyethylene glycol with a higher degree of polymerization. Further, the logarithm of the protein solubility is a linear function of the polyethylene glycol concentration. The process is reversible and the protein remains in its native form. An increase of the electrolyte (NaCl) concentration decreases the solubility of lysozyme in the presence and absence of polyethylene glycol. The effect can be explained by the screening of the charged amino residues of the protein. The solubility experiments were performed at two different pH values (pH = 4.0 and 6.0), where the lysozyme net charge was +11 and +8, respectively. Ion-specific effects were systematically investigated. Anions such as Br(-), Cl(-), F(-), and H(2)PO(4)(-) (all in combination with Na(+)), when acting as counterions to a protein with positive net charge, exhibit a strong effect on the lysozyme solubility. The differences in protein solubility for chloride solutions with different cations Cs(+), K(+), and Na(+) (coions) were much smaller. The results at pH = 4.0 show that anions decrease the lysozyme solubility in the order F(-) < H(2)PO(4)(-) < Cl(-) < Br(-) (the inverse Hofmeister series), whereas cations follow the direct Hofmeister series (Cs(+) < K(+) < Na(+)) in this situation.

FIGURE 1

Absorbance (λ = 340 nm; T = 25.0°C) during the PEG titration of lysozyme solution at pH = 4.0. Examples are shown for an initial protein solution c₃ = 190 g/L (solid symbols) and c₃ = 160 g/L (open symbols) in 0.20 M NaCl titrated with c₂ = 0.30 g/mL PEG-20,000.

FIGURE 2

Solubility curves of lysozyme at pH = 4.0 in 0.20 M NaCl, obtained by titration with c₂ = 0.60 g/mL PEG-3000 (circles), c₂ = 0.45 g/mL PEG-10,000 (triangles), and c₂ = 0.30 g/mL PEG-20,000 (squares). Lines represent the results of least squares fit of experimental data by Eq. 5.

FIGURE 3

Solubility curves of lysozyme at pH = 4.0 and 6.0 as obtained after titration with c₂ = 0.30 g/mL PEG-20,000 at various NaCl concentrations: 0.20 M (squares), 0.25 M (triangles), and 0.30 M (circles). Solid symbols correspond to pH = 4.0 and open to pH = 6.0. Lines are as for Fig. 2.

FIGURE 4

Solubility curves of lysozyme at pH = 4.0 in 0.20 M NaCl (squares), NaBr (circles, open circles after dialysis; see text), NaH₂PO₄ (diamonds), and NaF (triangles) obtained by titration with c₂ = 0.30 g/mL PEG-20,000. Lines are as for Fig. 2.

FIGURE 5

Solubility curves of lysozyme at pH = 4.0 in 0.20 M NaCl (squares), KCl (circles), and CsCl (triangles) obtained after titration with c₂ = 0.30 g/mL PEG-20,000. Lines are as for Fig. 2.

FIGURE 6

CD spectra before (solid line) and after (dotted line) precipitation of lysozyme for various protein concentrations (c₃). (From bottom to top) 0.57 g/L, 0.30 g/L, and 0.11 g/L. (Inset) ellipticity θ versus protein concentration at λ = 220 nm. Solid symbols correspond to ellipticity before and open after aggregation. The experiment was performed at pH = 4.0 in 0.20 M NaCl and PEG-20,000 (c₂ = 0.30 g/mL).