Lysozyme/Alginate Interactions: Structural and Thermodynamic Insights through ITC and SAXS.

Abstract

Coacervation and aggregation are distinct phase separation phenomena influenced by molecular properties and physicochemical conditions, such as pH and ionic strength. We investigate lysozyme (LYS)-alginate (ALG) mixtures at pH 7, focusing on the role of ionic strength in determining whether liquid-liquid phase separation (LLPS) or liquid-solid phase separation (LSPS) occurs. Using Isothermal Titration Calorimetry (ITC) and Small-Angle X-ray Scattering (SAXS), we find that a low salt (0-50 mM NaCl) induces compact fractal aggregates, while the intermediate salt (100-150 mM) yields coexisting or pure coacervates composed of larger swollen primary globules. At 200 mM NaCl, soluble complexes form instead of phase separation. ITC data reveal that both LSPS and LLPS are electrostatically driven, with the binding strength decreasing ∼50-fold from LSPS to LLPS conditions. These results demonstrate that phase behavior is tunable via ionic strength and that stronger interactions correlate with denser structures, highlighting distinct structural and energetic signatures for LSPS and LLPS.