Capillary electrophoresis methods for the determination of covalent polyphenol–protein complexes

Abstract

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.

Wine science in the metabolomics era

“…Furthermore, novel CE methods could be exploited to carry out metabolomic strategies. Thus, CE methods developed by pairing capillaries with different diameters with appropriate alkaline borate [40] could be useful to identify free proteins form covalently-bound protein-polyphenol complexes and monitor oxidation products to assess the traceability of wines. Additionally, the use of carbon nanotube-modified electrodes not only provides electrocatalytic properties, but also enhances signal stability and increases resistance to passivation for its application as an amperometric detector in the CZE separation of wine polyphenols [39].…”

“…The large number of applications of CE-MS has demonstrated its suitability for several -omics approaches [22,23]. In the recent bibliography, novel CE methods were applied to the analysis of wines, among other different fruit juices and beverages [39][40][41]. However, its use in the field of oenology for metabolomics applications to wine authentication is limited to a few studies.…”

An optimized method to extract poplar leaf proteins for two‐dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds

“…For many decades, it has been known that phenolics and quinones can participate in numerous reactions with proteins . These interactions can affect the electrophoretic properties of proteins or their activities . More importantly, however, oxidized phenolics are more hydrophobic and susceptible to aggregation and precipitation with proteins .…”

“…Although there is much information about the reaction of phenolic compounds with proteins, we found very few papers that demonstrated their effects on electrophoresis. At most, these studies only used gel‐shift assays to analyze the properties of the phenol–protein complexes themselves . To the best of our knowledge, only one experimental study confirmed that phenolic compounds may cause streaking of spots in 2DE gels .…”

Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation

“…However, if excess EGCg is added, nonspecific binding to the protein is initiated, leading to damage similar to that induced by glycation. The mechanism for damage includes the well-known formation of covalent bonds between the electron-dense B ring of the flavonoid and electrophilic groups on the protein such as amines or thiols [33,34], analogous to glycation reactions at these residues [9]. Increased carbonyl content is a consequence of the irreversible oxidative deamination of the polyphenol-protein adduct [35], while increases in fluorescence are attributed to the association of the protein with the highly aromatic polyphenol [33,34].…”