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.
INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|Laboratory of Research in Milk Products, Universidade Federal de Viçosa , BR-36570 Viçosa, Brazil.
Guilherme M
GM
Tavares
INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.
Thomas
T
Croguennec
AGROCAMPUS OUEST, Applied Mathematics Laboratory, F-35042 Rennes, France.
Sébastien
S
Lê
INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.
Olivia
O
Lerideau
INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.
Pascaline
P
Hamon
Laboratory of Research in Milk Products, Universidade Federal de Viçosa , BR-36570 Viçosa, Brazil.
Antônio F
AF
Carvalho
INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.|AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France.
Saïd
S
Bouhallab
D002151:Calorimetry / Q000379:methods; D005492:Folic Acid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007781:Lactoferrin / Q000737:chemistry*; D008956:Models, Chemical; D009994:Osmolar Concentration; D010316:Particle Size; D066329:Protein Aggregates; D011485:Protein Binding; D055672:Static Electricity; D013816:Thermodynamics
C490098:LTF protein, human; D066329:Protein Aggregates; D005492:Folic Acid; D007781:Lactoferrin