The inverse and direct Hofmeister series for lysozyme

Abstract

Anion effects on the cloud-point temperature for the liquid-liquid phase transition of lysozyme were investigated by temperature gradient microfluidics under a dark field microscope. It was found that protein aggregation in salt solutions followed 2 distinct Hofmeister series depending on salt concentration. Namely, under low salt conditions the association of anions with the positively charged lysozyme surface dominated the process and the phase transition temperature followed an inverse Hofmeister series. This inverse series could be directly correlated to the size and hydration thermodynamics of the anions. At higher salt concentrations, the liquid-liquid phase transition displayed a direct Hofmeister series that correlated with the polarizability of the anions. A simple model was derived to take both charge screening and surface tension effects into account at the protein/water interface. Fitting the thermodynamic data to this model equation demonstrated its validity in both the high and low salt regimes. These results suggest that in general positively charged macromolecular systems should show inverse Hofmeister behavior only at relatively low salt concentrations, but revert to a direct Hofmeister series as the salt concentration is increased.

Fig. 1.

(A) A dark field image from a linear array of 6 microcapillary tubes. Channels 1 and 6 were filled with polymer solutions and used as standards to calibrate the temperature gradient. Channels 2 through 5 were filled with 90.4 mg/mL lysozyme solutions at pH 9.4 with 0.10, 0.12, 0.14, and 0.16 M NaSCN, respectively. (B) A light scattering intensity curve taken from the lysozyme solution containing 0.10 M NaSCN. The cloud-point temperature was determined by drawing straight purple lines through the data during the transition and above the transition. The intersection of these lines is denoted by a dashed red vertical line, which is taken to be the cloud-point temperature. The region of the fluorescence micrograph that was used to obtain this linescan is denoted with a red line in A.

Fig. 2.

The cloud-point temperature of lysozyme as a function of anion type and concentration. All experiments were conducted with 90.4 mg/mL lysozyme in 20 mM Tris buffer at pH 9.4. The dashed lines are fits to the data points using Eq. 1.

Fig. 3.

(A) Residual cloud-point temperature data from Fig. 2 after subtracting the linear portion from the curves. (B) The residual cloud-point temperature data after removing the binding term. The dashed lines represent fits to the data points.

Fig. 4.

(A) Plot of partial molar volume of the anions vs. B_max. (B) Plot of the partial molar volumes of anions vs. the constant, b.

Fig. 5.

(A) Plot of surface tension increment values for the anions at the air/water interface vs. the constant, c. (B) Plot of polarizability values for the anions vs. the constant, c. Polarizability data are taken from ref. .