Salt-dependent complex formation in lysozyme-alginate mixture
Résumé
The associative interaction between oppositely charged macromolecules proceeds through either
liquid-liquid phase separation (LLPS) forming complex coacervates or liquid-solid phase separation
(LSPS) forming aggregates. In this work, we investigated the assembly of the basic protein lysozyme
(LYS) with the negatively charged polysaccharide alginate (ALG) at pH 7 under varying conditions
of mixing ratios, total concentrations, and ionic strengths.1
The droplet-based millifluidic device coupled with optical microscopy gave an extensive qualitative
analysis of the phase behaviour of the system by probing different experimental conditions.2 Grey
level analysis associated with the droplet millifluidic experiment allowed both the quantification and
the qualitative definition of phase separation. We constructed a three-dimensional phase diagram,
incorporating salt, LYS, and ALG concentrations as coordinates, offering a detailed depiction of
monophasic regions, liquid-solid and liquid-liquid phase separation domains, and areas of
coexistence of both solid and liquid phases. The thermodynamic characterization of the formation of
different LYS/ALG assemblies was carried out using isothermal titration calorimetry (ITC) where
distinct ITC profiles were associated with coacervation and aggregation. The interaction affinity (Ka)
for aggregation was three orders of magnitude higher than for coacervation, without significant
change in binding stoichiometry. Structural differentiation of various assemblies in the nanometer
range was achieved through small-angle X-ray scattering (SAXS) experiments. To gain deeper
insights into the mechanisms underlying both LSPS and LLPS processes, further investigation of
additional polyelectrolyte couples is needed, facilitating a more comprehensive understanding of
these phase separation phenomena.
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