Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG

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• 作者：Francis Canon ; Franck Pat茅 ; V茅ronique Cheynier ; Pascale Sarni-Manchado ; Alexandre Giuliani ; Javier P茅rez ; Dominique Durand ; Joaquim Li ; Bernard Cabane
• 刊名：Langmuir
• 出版年：2013
• 出版时间：February 12, 2013
• 年：2013
• 卷：29
• 期：6
• 页码：1926-1937
• 全文大小：635K
• 年卷期：v.29,no.6(February 12, 2013)
• ISSN：1520-5827

文摘

In the mouth, proline-rich proteins (PRP), which are major components of stimulated saliva, interact with tannins contained in food. We report in vitro interactions of the tannin epigallocatechin gallate (EgCG), with a basic salivary PRP, IB5, studied through electrospray ionization mass spectrometry (ESI-MS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS). In dilute protein (IB5) solutions of low ionic strength (1 mM), the proteins repel each other, and the tannins bind to nonaggregated proteins. ESI-MS experiments determine the populations of nonaggregated proteins that have bound various numbers of tannin molecules. These populations match approximately the Poisson distribution for binding to n = 8 sites on the protein. MS/MS experiments confirm that complexes containing n = 1 to 8 EgCG molecules are dissociated with the same energy. Assuming that the 8 sites are equivalent, we calculate a binding isotherm, with a binding free energy 螖渭 = 7.26RT_a (K_d = 706 渭M). In protein solutions that are more concentrated (0.21 mM) and at higher ionic strength (50 mM, pH 5.5), the tannins can bridge the proteins together. DLS experiments measure the number of proteins per aggregate. This number rises rapidly when the EgCG concentration exceeds a threshold (0.2 mM EgCG for 0.21 mM of IB5). SAXS experiments indicate that the aggregates have a core鈥揷orona structure. The core contains proteins that have bound at least 3 tannins and the corona has proteins with fewer bound tannins. These aggregates coexist with nonaggregated proteins. Increasing the tannin concentration beyond the threshold causes the transfer of proteins to the aggregates and a fast rise of the number of proteins per aggregate. A poisoned growth model explains this fast rise. Very large cationic aggregates, containing up to 10鈥?00 proteins, are formed at tannin concentrations (2 mM) slightly above the aggregation threshold (0.2 mM).