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 〈<i>Z</i>〉 = +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 (<i>C</i> = 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/<i>R</i>²) and ion-dipole (1/<i>R</i>⁴) 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.