Isothermal titration calorimetry study of epicatechin binding to serum albumin

Abstract

The interaction of epicatechin with bovine serum albumin (BSA) was studied by isothermal titration calorimetry. The binding constant (K) and associated thermodynamic binding parameters (n, ΔH) were determined for the interaction at three solution concentrations of BSA using a binding model assuming independent binding sites. These data show weak non-covalent binding of epicatechin to BSA. The interaction energetics varied with BSA concentration in the calorimeter cell, suggesting that the binding of epicatechin induced BSA aggregation. The free energy (ΔG) remained constant within a range of 2 kJ mol⁻¹ and negative entropy was observed, indicating an enthalpy driven exothermic interaction. It is concluded that the non-covalent epicatechin–BSA complex is formed by hydrogen bonding.

Introduction

Consumption of plants and plant products that are rich in polyphenols, such as cocoa, wine, tea and berries, has been related with protective effects against cardiovascular disease and certain forms of cancer [1], [2]. Polyphenols have been found to act as free radical scavengers and have been widely studied for their antioxidant activity in vitro [3], [4], [5]; however, questions remain concerning their in vivo activity, especially regarding their absorption, metabolism and bioavailability [6], [7], [8], [9], [10], [11]. Current literature suggests that factors such as protein binding may impair polyphenol absorption and bioavailability and even mask their antioxidant activity [12], [13], [14]. Non-covalent protein–polyphenol association is a well known phenomenon; however, it is only relatively recently that any considerable data from modern biophysical techniques (i.e., NMR spectroscopy, mass spectrometry, etc.) has been obtained in the area of how the structure of either the protein or the polyphenol may affect the interaction [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27].

Isothermal titration calorimetry (ITC) is an attractive approach for the study of biomolecular interactions such as the interaction of polyphenols with proteins. ITC sensitively measures changes in enthalpy during a titration experiment in which ligand is added to a protein solution in a calorimeter cell held under isothermal conditions [28]. It is a universal technique since enthalpy changes will occur during any interaction that leads to the formation of a complex, and only differential scanning calorimetry and ITC are able to directly determine interaction enthalpy. Furthermore, from a single experiment ITC allows the determination of association constant (K), stoichiometry (n), free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) of binding, and therefore provides a wealth of important data, even for low affinity interactions [29]. Principal advantages of ITC are that it can be used to characterise interactions in solution and without chemical modification or immobilisation of either interacting species. This sets the technique apart from surface plasmon resonance, which requires surface attachment, and fluorescence methods that often require labeling or are specific to proteins that contain a fluorophore that is accessible to a quencher. ITC can also be applied to systems where the complex formed is insoluble, as is often the case for protein–polyphenol systems. This is a distinct advantage over many solution based techniques, including capillary electrophoresis (CE), where complex insolubility can be problematic [24].

Our earlier research has demonstrated the potential for applying ITC analysis to the study of protein–polyphenol interactions through a study of the binding of two contrasting tannins, i.e., flexible tara gallotannins and more rigid ellagitannins (from myrabolan), to model proteins [21]. ITC data revealed differences in the binding data depending on the protein type, but could only be used for a qualitative study of binding due to the complexity of the tannin samples, which comprised a mixture of polyphenolic compounds of varying molecular weights. Here, we investigate the interaction of epicatechin as a model compound with bovine serum albumin (BSA). Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions; arguably the most important of these is as a depot and transport protein for a variety of compounds. BSA has been one of the most extensively studied of this group of proteins, particularly because of its structural homology with human serum albumin (HSA). (−)-Epicatechin ((2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol; Fig. 1) is a flavonoid, which are a group of natural products reported to have neuroprotective, cardioprotective and chemopreventive actions [30].