Tavares Guilherme M, Croguennec Thomas, Lê Sébastien, Lerideau Olivia, Hamon Pascaline, Carvalho Antônio F, Bouhallab Saïd
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.
Langmuir. 2015 Nov 17;31(45):12481-8. doi: 10.1021/acs.langmuir.5b02299. Epub 2015 Nov 3.
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.
在本文介绍的研究中，我们研究了叶酸（FA）与带正电荷的蛋白质乳铁蛋白（LF）在pH 5.5时的相互作用。我们发现结合常数Ka为10⁵ M⁻¹，化学计量比高达10摩尔FA/摩尔LF。在滴定实验过程中，形成的复合物的大小和电荷发生了变化。将离子强度提高到50 mM完全消除了等温滴定量热法（ITC）信号，这表明在观察到的放热结合过程中静电相互作用占主导。我们建立了一个理论模型来解释复合物的三相ITC图谱。我们的结果揭示了一个两步机制：FA/LF相互作用，随后是由此形成的复合物的自缔合。我们认为10个FA分子与LF结合形成饱和反应性复合物（FA₁₀/LF），该复合物进一步自缔合形成大小约为15 nm的聚集体。因此，蛋白质存在一个临界饱和度，超过该饱和度自缔合就会发生。我们在此展示了首批结果，这些结果提供了FA/LF络合-缔合热力学的全面细节。鉴于高化学计量比，每克LF可负载55毫克FA，我们认为FA/LF聚集体将是强化饮料中FA的有效载体。
