The role of charge regulation on casein-chitosan complexation at low pH

Abstract

The complexation between proteins and polyelectrolytes is fundamental to materials science and biology, yet the driving forces under non-ideal electrostatic conditions remain debated. Here, we systematically investigated the interaction between casein (CAS) and chitosan (CHI) at pH 5.5 and 3.0 using a combined experimental and theoretical approach. At pH 5.5, the oppositely charged macromolecules formed compact complexes through conventional electrostatic attraction (≈190–340 nm). A more intriguing behavior emerges at pH 3.0, where both CAS and CHI carry net positive charges yet still assemble into stable aggregates (≈340–370 nm). Spectroscopic analyses revealed that even under these conditions, CHI induced pronounced conformational and microenvironmental changes in CAS, including quenching of tryptophan fluorescence and secondary-structure remodeling, evidencing complex formation. To elucidate this counterintuitive phenomenon, we combined constant-pH Monte Carlo simulations with a semi-quantitative Kirkwood-Schumaker (KS) analysis. Our model quantified the mean charge of the representative αS1-casein as <Z> = +17.7 at pH 3.0, confirming strong electrostatic repulsion. However, we showed that the attraction is driven by the protein's significant charge regulation capacity (C = 3.45), resulting in a slightly shorter-range mesoscopic force that overcomes the repulsion. Within the KS framework, we also evaluated the ion–dipole (patch) contribution and demonstrated that, across the investigated pH range, it remains consistently smaller than the charge regulation term. The decisive role of this peculiar mechanism was confirmed experimentally: the complex dissociated upon the addition of salt, consistent with the screening of electrostatic interactions. Although both charge regulation (1/R2) and ion–dipole (1/R4) contributions are attenuated by the same exponential Debye screening, the longer-range nature of charge regulation makes it the dominant effect in the investigated pH range, thereby ruling out ion–dipole interactions as the primary driving force. This work provided a quantitative and mechanistic confirmation that charge regulation was the dominant driving force for protein-polyelectrolyte association on the “wrong side” of the isoelectric point, offering fundamental insights for the rational design of biomolecular complexes.