Experimental and Theoretical Studies on the Binding of Epigallocatechin Gallate to Purified Porcine Gastric Mucin

Abstract

Binding of epigallocatechin gallate (EGCG) to highly purified short side-chain porcine gastric mucin similar to human MUC6 type has been studied by ultraviolet-visible absorption spectroscopy (UV-vis), ultrafiltration isothermal titration microcalorimetry (ITC) and transmission electron microscopy (TEM). The thermodynamic equilibrium of EGCG binding to mucin has been quantitatively determined using ultrafiltration and high-performance liquid chromatography (HPLC)-UV/vis. The relationship suggests multilayer binding rather than simple Langmuir monolayer binding of EGCG. By combining the ultrafiltration and ITC data, the thermodynamic parameters of EGCG binding to mucin have been obtained. The binding constant for the first layer is about an order of magnitude higher than that of the consecutive multilayers. Negative entropy indicates multilayer of EGCG formed. Hydrogen bonding may be responsible for the multilayer formation. Increasing temperature resulted in a decrease in the binding affinity, further suggesting that hydrogen bonds dominated the interaction energy. A TEM micrograph of the EGCG-mucin complex revealed a monodispersion of blobs similar to pure mucin solution but with relatively bigger size (about twice). It is proposed that the EGCG-mucin binding process occurs by single and/or cluster of EGCG molecules driven to the surface of the two hydrophobic globules of mucin by hydrophobic interaction followed by hydrogen bond interaction between EGCG and mucin. Further adsorption of EGCG molecules onto bound EGCG molecules to form multilayers can also occur. This fits well with the observations that EGCG-mucin interaction followed a multilayer adsorption isotherm, the energy released is dominated by hydrogen bonds, and no large aggregates were formed.

Molecular and thermodynamic basis for EGCG‐Keratin interaction‐part I: Molecular dynamics simulations

“…The significance of the interactions with acidic amino acid side chains with EGCG has been also shown. The previously suggested 31 multilayer binding of EGCG to mucin via both hydrogen bonds and hydrophobic collapse is confirmed to be responsible for binding in the EGCG-keratin system at higher concentrations. In those simulations, EGCG molecules formed clusters which bind to keratin and spread over its surface.…”

“…We can, therefore, confirm that the formation of EGCG multilayer is in agreement with previous investigations of ultrafiltration and HPLC experiments in mucin-EGCG system. 31 …”

“…Previously, Zhao et al 31 showed that EGCG binds to gastric mucin by a combination of hydrophobic interactions and hydrogen bonding. Multilayer binding of polyphenol was suggested, and a decrease in binding affinity with increasing temperature was observed.…”

Molecular and thermodynamic basis for EGCG‐Keratin interaction‐part II: Experimental investigation

“…A variety of techniques were reported for the study of protein and phenolic compound interaction, such as equilibrium dialysis, 1,2 ultrafiltration, 3,4 fluorescence spectroscopy, 5 capillary electrophoresis, 6 and isothermal titration calorimetry (ITC). 4,7 Although most techniques are used to probe the kinetic and equilibrium parameters, the ITC method is used to determine the thermodynamic properties as well as equilibrium parameters of protein-ligand interactions.…”

Surface rearrangement of adsorbed EGCG–mucin complexes on hydrophilic surfaces

“…From the isothermal titration calorimetry data on EGCG-mucin systems it is known that the interaction of EGCG with mucin is characterised by weak binding [47]. These weak interactions may be insufficient to drive significant solidification during the aggregation of EGCG-mucin complexes, which otherwise would result in the loss of water.…”