The Research Of The Interaction Between Black Tea /green Tea Polyphenols And Human Salivary α-amylase By Enzyme Kinetic And Fluorescence Quenching

Objective: The aim of this study was to inspect the effectiveness and specificity of interaction between black/green tea tea polyphenols (TP) and human salivaryα-amylase (HSA) from the change of activity and molecular structure of HSA, through the enzyme kinetics and the fluorescence quenching experiments, to establish theoretical foundation to explain the physiological significance of the astringent effect and tea-induced pigmentation in mouth. Methods: The incident light at 540nm was used to measure the absorbance of maltose liberated by HSA in absence and presence of black/green tea inhibitors, then the reaction velocity was determined. Kinetic parameters (Vmax, Ks and Ki) were calculated using Lineweaver-Burk plots. The variance of parameters and the type of inhibition was determined by statistical analysis. The fluorescence intensity of HSA was detected at 285nm after dropwising different concentrations of black/green tea polyphenols solution. From the fluorescence spectra obtained, the Stern-Volmer (KSV), the bimolecular quenching (kq), the static quenching constants (KS), the apparent static quenching binding constant (KA), the apparent static quenching constant (K) and the othes related parameters were calculated, then the type of fluorescence quenching was decided. The variance of these parameters between black tea and green tea were analysed statistically. Result:There were significant differences between maximum velocity (Vmax) in the absence and presence of black/green tea inhibitor (P<0.05) and between the dissociation constants of enzyme-substrate (Ks) in the absence and presence of green tea inhibitor (P<0.05), the inhibitory patterns of black tea and green tea were noncompetitive and anticompetitive, respectively; Ks of black tea inhibitor was higher than the one of green tea inhibitor, through there was a reversal result of the dissociation constant of black tea inhibitor (Ki) (P<0.05). In the result of fluoresence quenching, the value of kq was larger than that of the bimolecular diffusion-controlled quenching constant (1×1010 M-1s-1), and there was a smallλem shift to lower wavelengths (a blue shift) with increasing TP concentration both in the black tea (5 nm) and green tea (18 nm) polyphenols solution; the type of quenching of black tea TP was demonstrated to be the static quenching, and the green tea was mainly the static quenching combined with the dynamic quenching; There was one binding sites(n=1)of black/green tea polyphenols with HSA. The quenching constants of KS, KA and K between black tea and green tea were significant differences; the KS and K of black tea were both larger than the one of green tea, through there was a reversal result of KA. Conclusion: The inhibitory of HSA by black tea were more effective than green tea, and the complex of black tea with HSA was more stable than green tea. The increasing hydrophobicity and the folding of protein molecules of HSA caused by green tea was stronger than black tea, and these led to more higher affinity of HSA to black tea than green tea. The results suggest that it is much easier to bring about the oral sensation of astringency and the stain deposites on the teeth surfaces for beverages of black tea.