Viscometric study of lysozyme solution with sugar and urea at various temperatures

1 Introduction

The native conformation of a protein is produced by a delicate balance between covalent bonds and noncovalent bonds such as hydrogen bonds, electrostatic interactions and hydrophobic interactions. Therefore, its conformation usually depends not only on temperature and pressure but also on the nature of the solvent, such as its polarity and dielectric constant. Hen egg white lysozyme is a well-known enzyme that acts as a glycoside hydrolase. This small globular protein consists of two functional domains located on each side of the active site cleft and contains both helices and regions of β sheet, together with loop regions, turns and disulfide bridges (Smith et al., 1993).

Very little attention has been paid to the viscosity of lysozyme aqueous solutions (Lefebure, 1982) and data of viscosity of lysozyme in mixed aqueous solutions are rare. Recently, attention has been paid, in particular, to the rich conformational variety of carbohydrates (Gabius, 2000; Hindley et al., 2005; Waris et al., 2001). Viscosity of egg-white lysozyme was measured in the presence of carbohydrate additives in reaction medium. These additives show a significant affinity for water. They depress water activity and increase the viscosity of the medium (Lamy et al., 1990). Solute–solvent interactions in aqueous solutions of the additives are characterized by B-coefficient.

The present work is a continuation of our research program on the thermodynamic studies on ternary systems (Siddique and Naqvi, 2010, 2011a). In this work viscosity measurements have been carried on sugars (d-glucose and maltose) and urea + aqueous lysozyme solutions (keeping the concentration of aqueous lysozyme solution (0.15 milli-molal) constant) at different temperatures (293.15, 303.15, 313.15 and 323.15 K) for different concentrations of sugar and urea to understand the increased or decreased stability of lysozyme in the presence of sugars and urea, respectively.

Heating of protein in solution can lead to aggregation, gelation, denaturation and thermal expansion, etc; depending upon the temperature range. The solute–solvent, solvent–solvent and solute–solute interactions in a protein solution undergo substantial changes upon exposure to different temperatures that bring about the observable physical change in the protein solution. As the thermal environment is altered, the Gibbs free energy, ΔG^∗ of the system changes, altering the physical state of the protein for which ΔG^∗ is minimized.

2 Materials and methods

Lysozyme (⩾99%) obtained from SIGMA–ALDRICH CHEMIE Gmbh Steimhein, Germany, was used for sample preparation. Sugars viz. d-glucose (⩾99%) and maltose (⩾99%) were obtained from Qualigans fine chemicals (a division of Glaxo Smith Kline Pharmaceuticals Limited, Mumbai). Urea crystal (⩾98%) extra pure was obtained from Merck Limited Worli, Mumbai. All solvents and chemicals were of analytical grade. These chemicals were used without further purification. The triply distilled water (with the specific conductivity of 1.29 × 10⁻⁶ Ω⁻¹ cm⁻¹) was used for making lysosome, sugars and urea stock solutions. All the solutions were stored in special airtight bottles to avoid exposure of solutions to air and evaporations.

The viscosity measurements were performed using an Ubbelohde-type capillary viscometer (Tanford, 1961). The working procedure is described elsewhere (Siddique and Naqvi, 2011b). The uncertainties in viscosity measurements have been found to be within ±0.003 mPa s. The densities required for the calculation of viscosity values of the solutions were taken from our earlier studies (Siddique et al., communicated) (unpublished data).

The triplicate reproducibility was established during the entire experimental work. The thermostatic paraffin bath (JULABO, Model-MD Germany) used during the measurements of density and viscosity was maintained at desired temperature (±0.02 K) for about 30 min prior to the record of reading at each temperature of study. The weighing was done on electronic balance (model: GR-202R, AND, Japan) with the precision of ±0.01 mg. The uncertainty in molal concentration values is found to be within 1.0 × 10⁻⁴ mol kg⁻¹.

3 Results

The experimental values of viscosity (η) are measured at different temperatures for lysozyme in aqueous and in sugar and urea solutions. These data are used to calculate the relative viscosity, (η_r) by the relation given below; (Jones and Dole, 1929; Tyrell and Kennerly, 1968; Devine and Lowe, 1971).

The B-coefficient values of the solute are obtained by the least-squares procedure. B-Coefficient is the measure of order or disorder introduced by the solute into solvent structure. This constant is specific and is an approximately additive property of ions of an electrolyte at a given temperature, although no satisfactory theoretical treatment has yet been given.

Viscosity data have also been used for the calculation of solute activation parameters (Pandey and Prakash, 1982). The free energy of activation (ΔG^∗) for viscous flow is given by Eyring viscosity equation (Eyring and John, 1969),

4 Discussions

Viscosities of sugars + aqueous lysozyme and urea + aqueous lysozyme systems are shown in Table 1 for different molalities of solute at different temperatures. The increase in concentration of solute increases the viscous behavior of the solution due to an increase in number of solute molecules, which causes more frictional resistance to the flow. But when we observed in case of urea in lysozyme solution, viscosity first decreases for lower concentration of urea (from 0.02 to 0.06 mol/kg) and then it gradually increases on further increase in concentration of urea. Therefore, we may conclude that at lower concentration of urea, its structure breaking effect is more pronounced while at higher concentrations it shows opposite behavior.

The increase in the concentration of solute in solution contributes positively to the viscosity B-coefficient. On the other hand, breaking of the solvent structure by solute causes a decrease in the viscosity. This contributes negatively to the B-coefficient. Thus, B-coefficient is the resultant of these two opposite forces (Mason et al., 1952). Therefore, the urea molecules exhibiting negative B-coefficient have been assumed to exert a structure breaking effect on the solvent while glucose and maltose exhibit effect on the solvent with positive B-coefficient and, thus, have structure-making effect on the solvent.

It has been observed (Table 2) that all the values of viscosity B-coefficient for saccharides are positive and in aqueous lysozyme solution, these values are greater for maltose than for glucose. B-Coefficient depends directly on size, shape and charge of the solute molecules, and maltose has two glucose units joined by α-1,4-glucosidic linkage. Therefore, the order is B (glucose) < B (maltose). It is noteworthy that the B (maltose) is not twice as large as that of is B (d-glucose), indicating that the formation of α-1,4-linkage reduces the structure making effects of saccharides.

According to Feakin's model (Feakins et al., 1974) greater the value of ΔG^∗, the greater is the structure making ability of solute. A perusal of Table 1 shows that ΔG^∗ increases with increase in temperature. This, thereby, indicates that the structure making ability of solute increases with temperature. Negative values of ΔS^∗ (Table 3) suggest that the attainment of transition state for viscous flow is accompanied by bond formation and increase in order.