Abstract
(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.
Highlights
Epigallocatechin 3-O-gallate (EGCG) (Figure 1A), a major catechin in green tea, exhibits antioxidant [1,2], antimutagenic [3], anticancer [4,5,6], antiallergic [7,8], and antiatherosclerotic [9,10] properties
We report EGCG fluorescence in four solvents, 1) EtOH, a protic solvent, 2) aqueous buffer (AB) at pH=7.0, as a model for the aqueous cytoplasmic environment, 3) DMSO, an aprotic polar solvent widely used for solubilization of waterinsoluble organic compounds in biomedical research, and 4) AN, an aprotic solvent widely used for liquid chromatography characterization of organic molecules
The rationale for this choice of solvents is that binding EGCG to a protein such as heat shock protein 90 (HSP90)[12] or to serum albumin[11] is likely to result in transition of EGCG from a predominantly aqueous environment to a less polar milieu which may result in dramatic changes in fluorescence[20]
Summary
EGCG (Figure 1A), a major catechin in green tea, exhibits antioxidant [1,2], antimutagenic [3], anticancer [4,5,6], antiallergic [7,8], and antiatherosclerotic [9,10] properties. We report EGCG fluorescence in four solvents, 1) EtOH, a protic solvent, 2) AB at pH=7.0, as a model for the aqueous cytoplasmic environment, 3) DMSO, an aprotic polar solvent widely used for solubilization of waterinsoluble organic compounds in biomedical research, and 4) AN, an aprotic solvent widely used for liquid chromatography characterization of organic molecules The rationale for this choice of solvents is that binding EGCG to a protein such as HSP90[12] or to serum albumin[11] is likely to result in transition of EGCG from a predominantly aqueous environment to a less polar milieu which may result in dramatic changes in fluorescence[20]. Being able to distinguish EGCG in these environments would provide an important tool for studying EGCG binding to proteins and offer the possibility of a direct binding assay using a target protein
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