Study On The Interaction Of Several Food Additives With Protein And DNA Based On Spectroscopy And Molecular Simulation

As an indispensable part of food industry,food additives play a significance role in prolonging the shelf life,improving the sensory indicators such as color,aroma and taste and enhancing the nutritional value of food.Food additives can be classified into synthesis and natural food additives according to their source.Synthetic food additives have wildly used in food industry due to their many advantages,such as stability and low production costs.However,the abuse of synthetic food additives can cause serious harm to human body,so their safety has been controversial.Different from synthetic additives,natural food additives are safe and possess some functional and nutritional properties.Biomacromolecules such as serum albumin and DNA,often are deemed as the targets of many small molecules like drugs,natural active ingredients and harmful chemicals,and they can also be used as models to investigate the toxicological or functional properties of food additives.Study on the binding mechanism of food additives with serum albumin and DNA is contribute to further understanding harmfulness of synthetic food additives and the function of natural food additives.In order to evaluate the potential hazards of synthesis food additives and promote the applied values of natural food additives in food processing and functional food development,four different food additives,quinoline yellow,tert-butylhydroquinone?TBHQ?,isoeugenol and protocatechuic acid were selected to investigated their binding mechanism with human serum albumin and DNA by fluorescence,ultraviolet-visible?UV–vis?absorption and circular dichroism?CD?spectroscopy combined with chemometrics algorithm multivariate curve resolution-alternating least squares?MCR-ALS?and molecular modeling analysis under simulation physiological conditions?pH=7.4?.The main contents and results of this paper were listed as follows:1.The binding mechanism of food colourant quinoline yellow?QY?with human serum albumin?HSA?and its effect on protein structural and functional properties were investigated through various spectroscopic methods and molecular docking analysis under simulation physiological conditions.It was found that QY could bind to HSA and form a HSA-QY complex,the binding constant was calculated to be on the order of 10⁴ L mol^-1,Hydrophobic interactions and hydrogen bonding were the main forces in the binding process.QY bound to the subdomain IIA of HSA which was corroborated by the molecular docking results.Decreases of HSA surface hydrophobicity and free sulfhydryl groups content indicated that QY caused a contraction of the peptide strand in HSA and hided the hydrophobic patches of the protein.The analysis of UV-vis absorption,CD spectra and three-dimensional fluorescence spectroscopy found that QY led to the microenvironmental perturbations around the fluorophores and secondary structure changes of HSA,the content of?-helix was increased along with a decrease of?-sheet.2.The binding properties of food antioxidant tert-butylhydroquinone?TBHQ?and its metabolite tert-butylquinone?TBQ?with calf thymus DNA?ctDNA?were investigated by UV-vis absorption,fluorescence and CD spectra methods,chemometrics algorithm,viscosity measurements,gel electrophoresis studies and molecular simulation.The concentration profiles and the pure spectra for the components?TBHQ/TBQ,ctDNA and TBHQ–or TBQ–ctDNA complex?obtained through analyzing the expanded UV-vis absorption data matrix by multivariate curve resolution-alternating least squares confirmed the QY-HSA interaction process,and the quantitative detection of the reaction process were successfully carried out.Analyses of fluorescence spectra indicated that both TBHQ and TBQ could bind to ctDNA and form complexes,and the binding constant between TBHQ and ctDNA was higher than that of TBQ with ctDNA.The results of AO,Hoechst33258displacement experiments and viscosity measurements suggested that TBHQ bound to ctDNA through intercalation binding,while TBQ interacted with ctDNA by groove binding mode.TBHQ could disturb the base stacking and double helix structure of ctDNA,and induce changes of DNA conformation from B form to A form,while TBQ had no obvious effect on DNA conformation.Gel electrophoresis results indicated that TBHQ and TBQ did not cause any cleavage in plasmid DNA,but the combination of TBHQ and Cu?II?together could induce synergistic DNA damage.3.The interaction of isoeugenol with ctDNA and its protective effect on DNA oxidative damage were investigated by multispectroscopic techniques,viscosity measurements,gel electrophoresis studies and molecular simulation under simulation physiological conditions.Analyses of UV-vis absorption and fluorescence spectra indicated that isoeugenol could bind to ctDNA with a binding constant on the order of10³ L mol^-1,which was mainly driven by hydrogen bonding and van der Waals forces.The results from the viscosity measurements,competitive displacement studies and CD analysis indicated that the binding of isoeugenol to ctDNA was in the groove mode and that the A-T base pairs in the minor grooves of ctDNA were the main binding sites for isoeugenol,which was supported by molecular docking analyses.In addition,isoeugenol was found to show a protective effect against hydroxyl-induced DNA damage,which may be explained by its good antioxidant activity.4.The inhibitory effect of natural food antioxidant protocatechuic acid on protein amyloid fibers and its possible inhibitory mechanism were studied by using human serum albumin as amyloid model.The results of ThT fluorescence assay indicated that procatechuic acid inhibited protein amyloid fibers in a dose-dependent manner,when the concentrations of procatechuic acid were 2.5×10^-44 mol L^-11 and5.0×10^-44 mol L^-1,the inhibition rates of protein amyloid fibers were 42.5%and59.5%,respectively.Transmission electron microscopy?TEM?also visually showed the inhibitory effect of protocatechuic acid on protein fibrosis.ANS fluorescence and CD spectra results suggested that procatechuic acid could significant decrease the exposure of hydrophobic amino acid residues on the surface of fibrosis protein and stabilize the?-helix structure of protein.Molecular docking results indicated that procatechuic acid interacted with human serum albumin through hydrophobic interactions and hydrogen bonding,and specifically bound to lysine?Lys?residues to maintain the conformational stability of protein,thus achieving the effect of inhibiting protein amyloid fibers.