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New Studies of epigallocatechin gallate (EGCG)

Epithelial mucus covers the external surface of mouth, gastric and intestine. Mucin glycoproteins are the major constituents of epithelial mucus.1 The interaction of saliva mucin with food/drink ingredients has an important effect on flavor and perception. Binding of polyphenols to saliva mucin is believed to be responsible for the sensation of astringency.2 Binding of polyphenol compounds to intestinal mucin has an important effect on nutrition and gut health.3 Epigallocatechin gallate (EGCG, Figure 1) is the most abundant polyphenol in green tea and has been associated with many medicinal and health benefits.4-7 Interactions of EGCG with food ingredients ,9-11 human serum albumin (HSA),12-14 proline-rich protein,15-17 and bovine mucin.18 A three-stage polyphenol-protein binding model has been proposed by Charlton et al19 for polyphenol binding to proline-rich protein. It was proposed that protein and polyphenols combine to form soluble  complexes. Such complexes aggregate to form colloidal particles, which  can further lead to sedimentation. Such three interaction between  polyphenol and glycosylated protein15,21,22 but understanding of the interaction is still limited. Pascal, et al.15 reported the effect of protein glycosylation on the aggregation of proline-rich protein and EGCG. They proposed that the threshold for aggregation must be related to protein concentration as well as to the EGCG/protein ratio. It is still not clear on which site EGCG binds to glycosylated protein and how strong the binding energy is between EGCG and protein. The thermodynamic equilibrium of EGCG binding to glycosylated protein is also not fully understood.

Phenolic compounds can interact with protein via either noncovalent interaction or covalent interaction.23 Covalent interaction takes place by oxidating the phenolic compounds to radicals and quinones.23 Very often, phenolic interaction with protein is noncovalent.24 Noncovalent interaction includes electrostatic interaction, van der Waals interaction, hydrogen bonds, hydrophobic interaction, and π bonds. Noncovalent interaction between protein and phenolic compounds can be studied by various techniques such as equilibrium dialysis (ED),25,26 ultrafiltration,27,28 ultraviolet-visible absorption spectroscopy (UV-vis),29,30 fluorescence spectroscopy,10,12 capillary electrophoresis,31 and isothermal titration micro-calorimetry (ITC).11,17,32 Using ED and ultrafiltration, the thermodynamic equilibrium of phenolic compounds binding to protein can be determined. However, phenolic compounds can also bind to the membrane used. Furthermore, the ED method is very time-consuming. The spectroscopy method measures the binding afinity in the environment of certain amino acid residues, while the ITC method provides the thermodynamic properties of enthalpy change of the interaction during a titration experiment. By fitting the enthalpy data into a proper binding model, the association constant (K), stoichiometry (n), free energy (G), and enthalpy (H) associated with the interaction can be obtained. However, ITC is a very delicate method and not suitable for both strong and weak interactions.23

In this paper, various measurement techniques are combined to investigate the binding of EGCG to purified porcine gastric mucin. Highly purified short side-chain porcine gastric mucin similar to human MUC6 type is selected. The mucin used is characterized as a highly glycosylated glycoprotein.33-35 Ultrafiltration method has been applied to quantify the thermodynamic equilibrium of EGCG binding to mucin. The associated thermodynamic properties of EGCG binding to mucin including the effect of temperature have been investigated using ITC. The microstructure and properties of the EGCG-mucin complex have been examined by transmission electron microscopy (TEM). The experimental data of ultrafiltration is best fitted by the Guggenheim-Anderson-deBoer (GAB) isotherm model, suggesting multilayer binding of EGCG to mucin. By combining the results of ultrafiltration and ITC, the thermodynamic properties of EGCG binding to mucin have been obtained. A model is proposed to describe the interaction between EGCG and mucin.

EGCG binding to mucin has been investigated by a combination of techniques of UV-vis absorption spectroscopy, ultrafiltration, TEM, and ITC. The UV-vis result supports that the carbonyl group present in the structure of EGCG is involved in the binding to mucin. Further NMR and IR studies could confirm this. The thermodynamic equilibrium of EGCG binding to mucin was determined by ultrafiltration, and the data is best fitted by GAB isotherm, suggesting multilayer EGCG binding to mucin rather than simple monolayer binding. The thermodynamic property of EGCG binding to mucin has been further characterized by ITC. The recorded enthalpy for EGCG-mucin binding is relatively low. The binding constant for the first layer is about an order of magnitude higher than the multilayers. Negative entropy indicates that a multilayer of EGCG formed during the binding process and also implies hydrogen bonding in the formation of the complex EGCG-mucin. Increasing temperature causes a decrease in the binding energy. This is a typical behavior of hydrogen bonds, further suggesting that the energy released during EGCG-mucin binding is dominated by that of hydrogen bonds. A TEM micrograph of the EGCG-mucin complex exhibited a similar monodispersion of blobs of the mucin-only system reported before,33-35 but about twice larger in radius. It appears that EGCG-mucin binding follows the steps of single and/or a cluster of a few EGCG molecules driven to the proximity of the hydrophobic heads of mucin by hydrophobic interaction at a molecular level followed by hydrogen bond interaction between EGCG and mucin at an atomic level. Free EGCG molecules can be also adsorbed onto already bound EGCG molecules to form multilayers. 

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