Spectroscopy Study On The Interaction Of Cephalosporins,Sunset Yellow,Tartrazine And Three Enzymes

In recent years,there are more and more studies on the interaction between small molecular substances and proteins.After entering the human body,small molecular substances will bind to the proteins in the body,so the study of the interaction between small molecular substances and proteins is of great significance to understand their transport,distribution and toxicity in the human body.Therefore,four systems of cefpiramine sodium（CPMS）-lysozyme（LYSO）,cefoxitin sodium（CFXS）-LYSO,sunset yellow（SY）-trypsin（TRP）and tartrazine（TTZ）-pepsin（PEP）are selected to study the interaction mechanism between cephalosporins,food pigments and common human enzymes.The specific research content is divided into the following six parts:Chapter one:The chapter mainly introduces the enzymes used in the experiment and the research methods in the experiment and looks forward to the research and development of small molecular drugs and enzymes.Chapter two:Under simulated physiological conditions,the interaction between CPMS and LYSO was studied by a variety of spectroscopy and molecular docking simulation techniques.The results showed that CPMS and LYSO formed a complex at 1:1 by static quenching,and the number of binding sites is about one.Through the discussion of thermodynamic parameters,it was known that the main forces of the binding system were hydrophobic force and hydrogen bond.UV absorption spectra and synchronous fluorescence spectra experiments showed that CPMS changed the conformation of LYSO.The results of molecular docking showed that the binding site of the two was located near the active center of LYSO,and the binding would affect the activity of the enzyme.The binding rate model of the system was established based on the data of spectral experiments.The drug binding rate W（Q）was 0.22%to 0.15%,and the protein binding rate was 81.86%to 88.10%.The combination of the two basically did not affect the efficacy of the drug,but it would affect the physiological function of the protein itself.Chapter three:Under the condition of pH=7.40,the binding of CFXS to tryptophan（Trp）and tyrosine（Tyr）residues in LYSO was studied by synchronous fluorescence spectroscopy.The results showed that both Trp and Tyr residues in LYSO participate in the binding reaction,and it can be inferred that CFXS quenched the fluorescence of Trp and Tyr residues by static quenching,and the fluorescence quenching ratio N_{SFQR（Trp）}of Trp residues was larger than that N_{SFQR（Tyr）}of Tyr residues,indicating that Trp residues contribute more to the binding reaction.According to the calculated thermodynamic parameters,the type of force was analyzed,and it was found that hydrophobic force and hydrogen bond were the main forces driving the binding of CFXS with Trp and Tyr residues.Molecular docking further illustrated this conclusion.The synergistic study of the system showed that the Hill coefficient n_H was about 1,indicating that there was no synergism between the drug molecule CFXS and the subsequent ligand.Through the establishment of the binding rate model,the protein binding rate and drug binding rate of Tyr residue and Trp residue of the binding system were calculated,and it was predicted that the binding effect of the two residues basically had no effect on the efficacy,but it would affect the physiological function of the protein.Chapter four:The binding mechanism of food pigment SY and TRP was studied by using a variety of spectral methods and molecular docking techniques.The results of UV experiments and fluorescence data analysis showed that the quenching of TRP by SY was based on the static quenching mechanism.The analysis of the thermodynamic parameters shows that the hydrophobic force and hydrogen bond are the main driving forces for the binding of SY and TRP.Synchronous fluorescence spectra and circular dichroism spectra showed that the conformation of TRP was changed by the combination of them.The results of molecular docking showed that SY was bound to the hydrophobic cavity of TRP and was surrounded by active amino acid residues histidine（His）His57,serine（Ser）Ser195 and aspartic acid（Asp）Asp102.Combined with the spectral experimental data,the binding rate model of the system was established,and the effect of SY on the physiological function of TRP was further predicted.Under the experimental conditions,the protein binding rate of SY and TRP system was10.80%⁶4.03%,indicating that SY would affect the digestive function of TRP.The distance r<7nm,indicating that there was a non-radiative energy transfer between SY and TRP.Chapter five:The interaction of food pigments TTZ and PEP was determined by fluorescence spectroscopy.The results of fluorescence analysis and UV absorption spectra showed that TTZ and PEP formed a 1:1 complex by static quenching,and the effect of TTZ on the conformation of PEP was studied by synchronous fluorescence spectroscopy and circular dichroism spectroscopy.The calculation results of thermodynamic parameters showed that hydrophobic interaction and hydrogen bond are the main forces of the binding system,and the results of molecular docking further proved this conclusion.The molecular docking method also revealed the best conformation and binding energy of TTZ and PEP.TTZ was binding in the hydrophobic cavity of PEP and was close to the catalytic active center of PEP,which changed the microenvironment of the active center residues.Chapter six:The interaction between CFXS and LYSO was studied under simulated physiological conditions by using the traditional fluorescence spectroscopy which focused on the fluorescence changes of proteins and the improved spectroscopy which focused on the fluorescence changes of drug small molecules by resonance light scattering.The results showed that the quenching mode of the binding system was static quenching,and the number of binding sites was 1.The binding constant K_a1 obtained by resonance light scattering method was slightly larger than that obtained by traditional fluorescence method,indicating that there was not only a"point-to-point"binding mode between CFXS and Tyr and Trp residues,but also a"point-to-side"binding mode with other amino acids.Finally,the rationality of the experimental results was verified by UV spectroscopy.