Proton-coupled protein binding: controlling lysozyme/poly(acrylic acid) interactions with pH.

Rational design of protein-polymer composites and their use, under the influence of the stimulus, for numerous applications requires a clear understanding of protein-polymer interfaces. Here, using poly(acrylic acid) (PAA) and lysozyme as model systems, the binding interactions between these macromolecules were investigated by isothermal titration calorimetry. The binding is proposed to require and be governed by "charge neutralization of the protein/polymer interface" and predicted to depend on solution pH. Calorimetric data show strong exothermic binding of lysozyme to PAA with a molar ΔH and TΔS values of -107 and -95 kcal/mol, respectively, at pH 7 and room temperature. Both ΔH and TΔS decreased linearly with increasing pH from 3 to 8, and these plots had slopes of -17.7 and -17.5 kcal/mol per pH unit, respectively. The net result was that the binding propensity (ΔG) was nearly independent of pH but the binding stoichiometry, surprisingly, increased rapidly with increasing pH from 1 lysozyme binding per PAA molecule at pH 3 to 16 lysozyme molecules binding per PAA molecule at pH 8. A plot of stoichiometry vs pH was linear, and consistent with this result, a plot of ln(average size of the protein/polymer complex) vs pH was also linear. Thus, protonation-deprotonation plays a major role in the binding mechanism. "Charge neutralization" of the lysozyme/PAA interface controls the binding stoichiometry as well as the binding enthalpies/entropies in a predictable fashion, but it did not control the binding affinity (ΔG). The pH dependence of lysozyme binding to PAA, demonstrated here, provides a stimuli-responsive system for protein binding and release from the polymer surface.