Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks.