Dynamic Monitoring Of The Interaction Of The Three Kinds Of Natural Pigments With Human Saliva And Histatin 5 By Surface Plasmon Resonance And Fluorescence Quenching

Objective: The aim of this study was to monitor the dynamic binding process of three kinds of natural pigments to human saliva and salivary histidine-rich proteins 5 (Histatin 5) by surface plasmon resonance(SPR) technique, to compare the effects of fluorescence quenching of Histatin 5 intrinsic fluorescence with different pigments, and to investigate the mechanisms of interaction between salivary proteins and pigments. Methods: In situ dynamic monitoring the binding process of three kinds of pigments(theaflavin, TF;cyanidin, Cy curcumin,Cur) to self-assembled monolayer biofilm of human saliva and Histatin 5 on SPR sensor surface in real time. The adsorption models of three kinds of pigments were decided by the correlation coefficient (R²) of Langmuir (L) and Freundlich (F) adsorption isotherm. The kinetic constants: Langmuir constant (K), maximal mass of adsorption (M_m), Freundlich constant (K_f), Freundlich intensity constant of adsorption (1/n), constant of association and dissociation rate (k_s and k_d), equilibrium constant of association and dissociation (K_A and K_D) were calculated, respectively. From the Histatin 5 fluorescence spectra obtained, the Stern-Volmer (K_SV), the bimolecular quenching (k_q), the apparent quenching (K_app), the dynamic quenching (K_D), the static quenching (K_S), the apparent static quenching (K), the static quenching combination(KLB), the apparent static quenching combination(KA) constants and the number of the combined sites (n) were calculated, respectively. The datas was analyzed by AVNOA, SNK-q test and paired t-test The level of significance was defined asα=0.05. Result: Both of Langmuir and Freundlich models can be used for describing the binding processes of Cy/WS and TF/H5. Freundlich model is more suitable for describing the binding processes of TF/WS,Cur/WS,Cy/H5 and Cur/H5. Both of the intensity extents of interaction between pigment/ H5 and WS/ H5 are Cur> TF and Cy, Mm are TF>Cur>Cy, Kf are TF>Cur and Cy,then Cur is closed to Cy. Both of the types of fluorescence quenching of TF/H5 and Cur/H5 are dominant static quenching reaction, accompany with the dynamic and distance quenching, and the type of Cy/H5 is static quenching reaction. The number of the combined sites of the interaction between pigments and Histatin 5 are all about 1. It means that the biomolecular interaction between pigments and Histatin 5 is one to one. During the process of fluorescence quenching, Cy can change the molecular conformation of Histatin 5 through increasing hydrophilic and decreasing hydrophobic of microenvironment of fluorophore. Fluorescence quenching extent of pigments/H5 is Cy>TF and Cur (P<0.05), TF and Cur are at the same level(P>0.05). Conclusion: Different kinds of pigments have the different affinity with human salivary proteins, flavonoids(TF and Cy) have the higher affinity than curcuminoid. Hydrogen bonding, electrostatic and hydrophobic interaction are important driving forces for salivary proteins-pigments interaction.