A study of alcohol and temperature effects on aggregation of β-lactoglobulin by viscosity and small-angle X-ray scattering measurements

Small-angle X-ray scattering (SAXS) of aqueous solutions of methanol and 2,2,2-trifluoroethanol (TFE) of β-lactoglobulin (β-Lg) in concentrations of 30 and 63 mg/mL was measured as a function of alcohol concentration in a temperature range of 298-363 K to investigate the effects of alcohol and temperature on aggregation of the protein. The viscosity of aqueous solutions of methanol, ethanol and TFE of β-Lg in a concentration of 2 mg/mL was also measured as a function of alcohol concentration at an ambient temperature. The relative viscosity, η / η0, where η and η0 are the viscosities of the protein solutions and the solvents, respectively, showed a maximum at around 50%, 40% and 20% of the methanol, ethanol and TFE solutions, respectively. This finding suggests that the aggregation of β-Lg of a low concentration occurs at the specific alcohol concentrations. The SAXS data showed that the aggregation of β-Lg in terms of the aggregation number and the radius of gyration initiates at an alcohol concentration of ~ 40% for the methanol solutions and ~ 10% for the TFE solutions and increases with alcohol concentration at ambient temperature. Furthermore, the temperature of thermal denaturation of β-Lg decreased by ~ 30 K for the TFE solution and by ~ 20 K for the methanol solution, compared with the case for water (348 K). Thus, the addition of TFE promoted the thermal denaturation of the protein more effectively than methanol. The aggregation structure of the thermally denatured protein was independent of type and concentration of alcohol in the concentrated protein solutions. Furthermore, the fractal dimension of the aggregates was ~ 2.6, indicating a three dimensional network structure. However, in the diluted protein solutions, the aggregate structure after the thermal denaturation was likely to be a low cross-linked network gel in the methanol solution, which was similar to that in water. On the other hand, more complex aggregates tended to form in the TFE solution.