Effect of Polypeptide Complex Coacervate Microenvironment on Protonation of a Guest Molecule

Complex coacervate droplets formed by the liquid-liquid phase separation of polyelectrolyte solutions capture several important features of membraneless organelles including their ability to accumulate guest molecules and to provide distinct microenvironments. Here, we examine how polyions in complex coacervates can influence localized guest molecules, leading to a shifted protonation state of the guest molecule in response to its electrostatic environment. A fluorescent ratiometric pH indicator dye was used as a model guest molecule able to report its protonation state in the coacervate phase. Experimentally observed differences in dye-reported local apparent pH inside versus outside of coacervate droplets were largest for polyion pairs having lower salt stabilities and/or larger polyion length mismatch, which we attribute to the relative concentration of open sites on polyions within the coacervates based on theoretical calculations. Using the transfer matrix method, we confirmed that theoretical phase diagrams and critical salt stabilities generated for each polyion pair were consistent with experimental turbidity measurements and estimated the amount of available binding sites on polyions for guest molecules. We conclude that dye molecules likely experience an effective pKa shift due to interactions with coacervate polyions rather than reporting directly on local proton concentrations. Such a local pKa shift can also be anticipated for other guest molecules having protonatable groups, including, for example, many metabolites, ligands, and/or drug molecules that partition into coacervates or membraneless organelles based on ion pairing interactions.