Spectroscopic Study On The Isomerization Of Resveratrol And Polydatin And Their Interaction With Protein

Fluorescence spectroscopy is an effective method to study molecular conformation,and provide a large number of parameters about molecular bonding and structure nature of the concerned materials.UV-vis absorption spectroscopy is a simple and direct tool for measuring the changes in the structure of the molecular.In situ attenuated total reflection infrared spectroscopy(ATR) can be used to explore a chemical or biological reaction at the molecular level by acquiring real time information about molecular structure of related species in non-destructive and non-invasive situation.In this thesis,fluorescence spectroscopy,UV-vis absorption spectroscopy and in situ attenuated total reflectance Fourier transform infrared spectroscopy have been used to study the interaction between protein and resveratrol or polydatin,the photo-isomerization and the mechanism of isomerization of resveratrol or polydatin.1.The recent researches using fluorescence spectroscopy,UV-vis absorption spectroscopy and attenuated total reflectance Fourier transform infrared spectroscopy(ATR-FTIR) have been briefly reviewed.2.The interaction between resveratrol and transferrin(Tf) was investigated using fluorescence spectroscopy,attenuated total reflectance Fourier transform infrared spectroscopy(ATR-FTIR) and UV-vis absorption spectroscopy at physiological pH(pH=7.0).The effects of fatty acid on the interaction between resveratrol and Tf were also investigated.The solubility,photostability and thermostability of resveratrol were detected both in the present and absence of Tf.It was confirmed that the intrinsic fluorescence of resveratrol and Tf were quenched with a static quenching mechanism by Tf and resveratrol, respectively.1:1 complex was formed through interactions of resveratrol with Tf.The binding constant of the complex was obtained in different ways.Hydrophobic and van der Waals forces played a major role in the interaction of Tf with resveratrol.The shortest distance(r=3.39 nm) between Tf and resveratrol was obtained according to F(o|¨)rster energy transfer theory.Synchronous fluorescence and infrared spectroscopy results suggested that the interaction between resveratrol and Tf leaded to changes in the conformation and secondary structure of Tf.Fatty acid exhibited both competitive and cooperative effects on the interaction of resveratrol with Tf.A slight increase in the photostability,thermostability of resveratrol and a significant increase in the hydrosolubility of resveratrol were observed while it was bounded to Tf.3.Binding reaction of polydatin with Bovine serum albumin(BSA) or lysozyme were studied by steady fluorescence spectroscopy in physiological conditions.The results show that self-association of polydatin possibly occurred at high concentrations.Polydatin had a strong quenching effect on BSA and lysozyme fluorescence.Static quenching and non-radiation energy transfer could be the mechanisms for the fluorescence quenching.According to Lineweaver-Burk equation and double logarithmic equation,the apparent binding constants,the numbers of binding sites of polydatin-BSA and polydatin-lysozyme were estimated,which were K_BSA=5.90×10⁴, n_BSA=0.997,K_lysozyme=3.86×10⁴,n_lysozyme=1.01,respectively. Fluorescence anisotropy suggested that the binding location of polydatin with BSA could be the site I of BSA,while sychronous fluorescence indicated that the binding site for polydatin in lysozyme could be the tyrosine of lysozyme.Hydrophobic interaction and hydrogen bond played a major role in the polydatin-BSA and polydatin-lysozyme interaction,respectively.According to F(o|¨)rster theory,the binding distance of polydatin-BSA was obseved to be shorter than polydatin-lysozyme.These results suggested that the polyphenol-protein interaction depend on the structure of protein.4.In situ ATR-FTIR was used to study the photoinduced isomerization of trans-polydatin.The UV-vis absorption spectroscopy studies showed that the wavelengths of ultraviolet radiation,irradiation time,and the pH values affected the isomerization speed,while the concentration of trans-polydatin showed mildly effect.The result obtained from the in situ ATR-FTIR indicated that vibration of C=C,=C-H,methyleneand substitution of benzene rings in trans-polydatin molecule changed during the process of UV irradiation.Furthermore,it is observed that after binding to BSA,the rate constant of isomerization decreased, indicating the photostability of trans-polydatin improved.The protective action of BSA against trans-polydatin damage in UV spectrum was related to the BSA concentration and ionic strength.