Pascaline Hamon

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.

Structure and Dynamics of Heteroprotein Coacervates.

Under specific conditions, mixing two oppositely charged proteins induces liquid-liquid phase separation. The denser phase, or coacervate phase, can be potentially applied as a system to protect or encapsulate different bioactive molecules with a broad range of food and/or medical applications. The optimization of the design and efficiency of such systems requires a precise understanding of the structure and the equilibrium of the nanocomplexes formed within the coacervate. Here, we report on the nanocomplexes and the dynamics of the coacervates formed by two well-known, oppositely charged proteins β-lactoglobulin (β-LG, pI ≈ 5.2) and lactoferrin (LF, pI ≈ 8.5). Fluorescence recovery after photobleaching (FRAP) and solid-state nuclear magnetic resonance (NMR) experiments indicate the coexistence of several nanocomplexes as the primary units for the coacervation. To our knowledge, this is the first evidence of the occurrence of an equilibrium between quite unstable nanocomplexes in the coacervate phase. Combined with in silico docking experiments, these data support the fact that coacervation in the present heteroprotein system depends not only on the structural composition of the coacervates but also on the association rates of the proteins forming the nanocomplexes.

How the presence of a small molecule affects the complex coacervation between lactoferrin and β-lactoglobulin

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins β-lactoglobulin (β-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with β-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of β-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached.

pH- and ionic strength-dependent interaction between cyanidin-3-O-glucoside and sodium caseinate

Understanding the mechanism of interaction between food proteins and bioactives constitutes the preliminary step to design food grade nanocarriers. We investigated the interaction between cyanidin-3-O-glucoside (C3G), and 20nm-sized sodium caseinate nanoparticles (NaCas) at pH 7 and pH 2 by fluorescence spectroscopy and dynamic light scattering. The characterization of the C3G-NaCas interaction indicated that the fluorescence quenching mechanism was predominantly static. C3G interacted with two sets of binding sites with association constants Ka of 106 and 105M-1. Electrostatic interactions dominated at pH 7, while hydrophobic effects were the main force at pH 2. Interestingly, the two sets of binding sites were discriminated by ionic strength at pH 7. The binding of C3G slightly modified the average diameter of NaCas nanoparticles without alteration of its surface charge suggesting a complexation of C3G molecules in the internal casein structure. Thus, NaCas constitutes a putative nanocarrier for anthocyanins in new functional foods.