Study On The Interaction Between Several Food Additives And Protein/DNA

The interacton mechanism between some synthetic food additives with serum albumin/ctDNA were studied in this thesis using multispectral technology, such as, fluorescence, Ultraviolet-visible (UV-vis) absorption, circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). These studies have important theoretical significance for further understanding toxic mechanism of artificially synthesized food additives.The main contents and conclusions in the thesis are summarized as follows:1. The structure, physiological function and biological characteristics of serum albumin and DNA were briefly stated at first in this chapter. And then, the research status and methods of bingding with small molecules were summarized.2. The binding mechanism of maltol with bovine serum albumin (BSA) under simulated physiological conditions was investigated by fluorescence, UV-vis, CD and FT-IR spectroscopy. The results suggested that the fluorescence quenching of BSA by maltol was a static procedure by forming new complex. The enthalpy change (ΔH) and entropy change (ΔS) were119.2±0.1kJ·mol-1and757.0±0.4J·mol-1·K-1using van’t Hoff equation, respectively, indicated that hydrophobic interaction played a predominant role in the interaction of maltol with BSA. The binding distance between maltol and BSA was3.01nm based on the Forster theory of non-radioactive energy transfer. The competitive experiments of site markers revealed that the binding of maltol to BSA mainly took place in subdomain IIA (site I). Moreover, the results of UV-vis, synchronous fluorescence, CD and FT-IR spectra indicated that the maltol could affect the polarity of microenvironment and change the secondary structure of BSA.3. With human serum albumin (HSA) as the model protein, the interaction mechanism of amaranth with HSA and conformational changes of HSA by amaranth in Tris-HCl buffer (pH7.4) were investigated by multispectral technology. Results obtained from fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure and the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth using ANS (8-anilino-l-naphthalenesulfonic acid) as a hydrophobic probe. The protein denaturation and competitive experiments suggested that the binding site for amaranth on HSA was site I. The results of CD and FT-IR spectra showed that binding of amaranth to HSA changed the conformation of HSA, decreased the content of a-helix, which will further impair the stability of HSA.4. The binding properties of butylated hydroxytoluene (BHT) with calf thymus DNA (ctDNA) in Tris-HCl buffer (pH7.4) were investigated using ethidium bromide (EB) as a fluorescence probe by UV-vis absorption, fluorescence, CD, and FT-IR spectroscopy along with ctDNA melting studies and viscosity measurements. It was found that the binding of BHT to ctDNA could decrease the absorption intensity of ctDNA, significantly increase melting temperature and relative viscosity of ctDNA. Moreover, the competitive binding studies showed that BHT was able to displace EB from the bound ctDNA-EB complex. All the experimental results indicated that the binding mode between BHT and ctDNA was an intercalation. Further, the CD and FT-IR analysis revealed that BHT was more prone to interact with adenine and thymine base pairs, and no significant conformational transition of ctDNA occurred.5. The interaction between food preservative, sodium benzoate (SB), and ctDNA in simulated physiological buffer using acridine orange (AO) dye as a fluorescence probe, was investigated by UV-vis absorption, fluorescence and CD spectroscopy along with DNA melting studies and viscosity measurements. An expanded UV-vis spectral data matrix was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The equilibrium concentration profiles and the pure spectra for SB, ctDNA and ctDNA-SB complex were simultaneously obtained. The results indicated that SB could bind to ctDNA and form ctDNA-SB complex. Moreover, SB was able to quench the fluorescence of ctDNA-AO complex through a static procedure, SB could increase increase melting temperature and relative viscosity of ctDNA and change the CD spectra of ctDNA, indicating that an intercalative mode between SB and ctDNA occurred.6. The interaction mode between food dye, indigo carmine (IC), and ctDNA in buffer of pH7.4was estimated by parallel factor analysis (PARAFAC). The three-way synchronous fluorescence spectra data obtained from the interaction between IC and ctDNA-EB were resolved by PARAFAC, and the concentration information and the pure spectra for IC, EB and ctDNA-EB at equilibrium were simultaneously extracted. PARAFAC analysis demonstrated that the intercalation of IC molecules into DNA proceeded by substituting for EB in the DNA-EB complex, which could be supported by ds/ss ctDNA quench experiments, melting studies and viscosity measurements.