Heteroprotein complex coacervation of lactoferrin and osteopontin: Phase behaviour and thermodynamics of formation

This research describes the formation of reversible heteroprotein complex coacervates between the bioactive milk proteins lactoferrin (LF) and osteopontin (OPN) within specific combinations of pH (∼4–6), ionic strength (≤30 mM added NaCl), protein stoichiometry (LF:OPN mass mixing ratios of ∼ 2–8) and total protein concentration (≤∼8% w/v). Electrostatic attraction between these proteins is possible across a wide range of pH values due to the acidic isoelectric point of OPN (∼ pH 3.5) and the basic isoelectric point of LF (∼ pH 8.7). Thermodynamic analysis of the OPN-LF interaction indicated that the protein-protein binding had enthalpic and entropic contributions which resulted in liquid-liquid phase separation given the appropriate combination of intrinsic and extrinsic conditions. The complex coacervates were found to be a concentrated protein phase (∼27% protein, w/w) which was highly hydrated (>70% moisture, w/w) and occurred spontaneously within LF: OPN ratios naturally found in human milk. The structure, biological functionality, and applications of heteroprotein complexes and/or complex coacervates between LF and OPN will be explored in separate work.