Dielectric behavior of lysozyme and ferricytochrome-c in water/ethylene-glycol solutions

Abstract

This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.

FIGURE 1

Increment of permittivity (ɛ′ − ɛ₀₀) (a) and relaxation dielectric loss ɛ″_d (b) of a solution of lysozyme in water/ethylene-glycol mixture. Fractional concentration of cosolvent 0.91 w/w. In the plot of ɛ″_d, the beginning of relaxation of the cosolvent is evident. Solid line is a Cole-Cole fit.

FIGURE 2

Ratio between solvent viscosity and relaxation time of solute for lysozyme in water/glycerol (a) and water/ethylene-glycol (b) mixtures as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted line for visual aim.

FIGURE 3

Hydrodynamic radius (a) and electric dipole moment (b) of lysozyme in water/ethylene-glycol mixtures as function of weight fraction of cosolvent. Calculations on the basis of the dielectric parameters of Table 1, obtained fitting the experimental data with one Cole-Cole dispersion. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted lines for visual aim.

FIGURE 4

Circular dichroism spectra in the far-UV (a) and near-UV (b) in water (solid line) and in water/ethylene-glycol solutions (dotted line). Fractional concentration of cosolvent 0.61 w/w.

FIGURE 5

Hydrodynamic radius (a) and electric dipole moment (b) of cytochrome-c in water/ethylene-glycol mixtures as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted lines for visual aim.

FIGURE 6

Molar fraction of open lysozyme molecules calculated by Eq. 4 in the text (a) and average electric dipole moment of lysozyme in water/ethylene-glycol mixtures, calculated considering the transition of the protein from native to open state (see text) (b) as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted line for visual aim.