Study On The Reaction Mechanism Of Tartrazine And Pioglitazone Hydrochloride With Three Proteins By Spectroscopy

In recent years,the study of the interactions between small molecules and proteins has become a hot topic.In this paper,pioglitazone hydrochloride-transferrin,pioglitazone hydrochloride-pepsin and pioglitazone hydrochloride-trypsin,tartrazine-trypsin were used as subjects.The four systems were studied by fluorescence spectroscopy,synchronous fluorescence spectroscopy,UV-Vis spectroscopy,circular dichroism spectroscopy and molecular docking technique.The research content was divided into the following sections:Chapter one:In this paper,several spectral methods and molecular docking simulation techniques used in the study of drug-protein interaction are reviewed,and the development of drug-protein research field is described and prospected.Chapter two:The interaction between pioglitazone hydrochloride（PGH）and tryptophan（T-Trp）residues and tyrosine（T-Tyr）residues in transferrin（TF）were investigated using synchronous fluorescence spectroscopy at different temperatures（298,310 and 318 K）.From K_a,n and thermodynamic parameters,it was shown that the stable compound of 1:1 was formed by electrostatic force interaction between PGH and T-Trp residues,PGH and T-Tyr residues.The binding degree between PGH and T-Trp residues was higher than that between PGH and T-Tyr residues.At 310 K,the fluorescence quenching ratio number(N_SFQR)of T-Tyr residues and T-Trp residues were 48.45%and 51.55%,respectively.That is,at the amino acid level,we can further understand the degree of fluorescence quenching of different amino acids in the protein when the drug interacts with the protein.The large N_SFQR value indicates that the combined drug has a large effect on the drug efficacy.At 310 K,binding rate of PGH andT-Tyrresidueswas55.60%⁷3.82%andcombinemodelwas W=-0.0315R²-0.1520R+0.7385.The value of Hill’s coefficients were greater than 1,which suggested there was a positive cooperativity between PGH and subsequent ligands.The r<7nm indicated that the static fluorescence quenching of T-Tyr residues and T-Trp residues by PGH was also a non-radiation energy transfer process.The results of molecular docking were consistent with that of experimental calculation.Chapter three:In order to study the effect of the interaction between drugs and pepsin（PEP）on the pharmacodynamics and digestive ability of stomach,the spectral determination was established under simulated physiological conditions.At 298 K,310 K and 318 K,the interaction mechanism between pioglitazone hydrochloride（PGH）and PEP was studied by fluorescence,synchronous fluorescence and molecular docking simulation.The results shown that a stable 1:1 complex was formed between PGH and PEP by electrostatic force and hydrogen bonding in a static quenching manner.Tyrosine residues（Tyr）and tryptophan residues（Trp）in PEP were involved in the reaction.The binding of PGH-PEP had positive cooperativity effect on the subsequent ligands.The results shown that when patients took PGH 15 mg⁴5 mg,the drug binding rate in gastric juice was 0.0013%⁰.0032%,which was very small,that is,the combination of PGH and PEP had little effect on PGH.The binding rate of PEP was 69.76%⁸7.37%,that is,PGH reduced the free PEP by 69.76%⁸7.37%,indicating that PGH would affect the digestive function of the patients.Molecular docking technique showed that the best binding site between PGH and PEP was located at the catalytic active site of PEP,and the interaction between them changed the microenvironment of amino acid residues in PEP catalytic active center.Chapter four:Pioglitazone hydrochloride（PGH）quenched the fiuorescence of trypsin（TRP）by static quenching.The microenvironment of Trp and Tyr residue was both changed and hydrophobicity decreased.The K_a,n andΔG,ΔH,ΔS of PGH-TRP indicated that the PGH and TRP formed a stable complex of 1:1 via hydrogen bonds and hydrogen interaction.According to the site competitive experiment,the main binding position between PGH and TRP was the hydrophobic pocketⅠof TRP.Binding rate of PGH to protein in 298 K is64.95%⁸5.03%and combine model is W=-0.08684R²+0.1048R+0.8503.Chapter five:The extent to which drugs combine with trypsin（TRP）is influenced by the interaction of tartrazine（TTZ）and TRP,which may cause effects on the efficacy of the drug.Therefore,the interaction of TTZ and TRP is investigated by methods of spectrometry in this paper.The binding rate of TTZ to TRP was 80.95%⁹5.71%at 310 K.The effect of TTZ on TRP structure was studied by synchronous and circular dichroism.The results showed that the binding of TTZ and TRP induced the conformational change of TRP,and quenched the intrinsic fluorescence in TRP.The results of molecular docking revealed that TTZ were located in the catalytic active site of TRP,that is,most drugs bind to TRP here,which further illustrates the competitive relationship between TTZ and drugs when binding to proteins,and are consistent with that of experimental calculation.