Calcium binding to β -lactoglobulin. Effects of pH and ionic strength

Calcium binding to native β-lactoglobulin (β-Lg) was quantified to clarify its role in dairy-relevant phenomena. Binding was measured across dairy relevant conditions, pH (5.5-7) and ionic strength (∼35-400 mmol/L) using equilibrium dialysis and inductively coupled plasma mass spectrometry (ICP-MS), with molecular dynamics simulations providing qualitative insight into binding motifs. Experimental data show that calcium binding plateaus scales linearly with intrinsic protein charge, consistent with site-specific association to Asp/Glu carboxylate groups rather than long-range electrostatics. Competitive binding by sodium ions was directly observed and explains discrepancies amongst reported binding constants. A deliberately empirical, low-parameter competitive binding model was developed for screened, dairy-relevant ionic strengths, reconciling literature values by accounting for co-ion competition and ionic strength effects. The model predicts ≤0.5 calcium ion per β-Lg monomer under milk-like conditions. The study does not model denatured state binding during heat treatment, with conclusions limited to regimes where long range electrostatics are suppressed.