A small angle X-ray scattering study comparing protic ionic liquid solvent effects on the solution behaviors of hen egg white and human lysozyme

Abstract

Proteins demand customized solvents for maintaining their stability. However, the specific impact of different ions on different proteins remains unclear. This study explores the effects of protic ionic liquid (PIL)-based solvents on two structurally similar proteins, namely hen egg white and human lysozyme. By employing size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and capillary SAXS, the effects of protein concentration (3 to 60 mg/mL), IL type (five PILs) and concentration (1, 10 and 20 mol% with water) and protein type on the aggregation behavior and radius of gyration (Rg) of the two lysozymes were studied. HEWL was mostly monomeric in buffer and solubilized beyond 20 mg/mL, while HL displayed slight aggregation in buffer but had increased aggregation at over 10 mg/mL. Further capillary SAXS data show that both proteins became more aggregated at concentrations exceeding 20 mg/mL and over 1 mol% PILs. The nitrate anion generally showed the “salting in” effect, while formate and glycolate anions induced the “salting out” effect on the two proteins. Overall, anion selection and protein solution behaviors at protein concentration of over 20 mg/mL and at 20 mol% ILs can be critical for maintaining the lysozyme stability.