Study On The Luminescence Behavior And Application Of Luminol-model Protein-small Molecule Ligands

Protein has many important functions in the biological activities,and the study of protein-small molecule interaction has become one of concern hot topic in chemistry,life sciences,clinical medicine and other areas.In this work,by luminol as luminescent probes,based on luminol-model protein system were established using flow injection chemiluminescence(FI-CL),as a basis to explore the interaction of lysozyme with lanthanide ions,the interaction of myoglobin with dibutyl phthalate,and its analytical application made a further study.The content of this thesis included two parts,as follows:Part I:ReviewChapter 1 IntroductionIn recent years,the interaction of protein with small molecules has become a hot spot in the fields of chemistry,biology and clinical medicine.Through researching the interaction,the study were analysis from binding parameters,binding sites,binding site numbers,the conformational changes of protein,thereby contributing to set forth the mechanism of protein and small molecule in many aspects,and it has become more practical significance.Many methods have been utilized to investigate this hot topic,such as,fluorescence spectroscopy,UV-visible absorption spectroscopy,circular dichroism spectroscopy,nuclear magnetic resonance spectroscopy and chemiluminescence.As common model proteins,bovine serum albumin,lysozyme,myoglobin and catalase are widely applied to the study of protein-small molecule interaction.Part Ⅱ:Research reportsChapter 2 Constructing Luminol-Protein system and studying the interactionThe interaction behaviors of bovine serum albumin(BSA),lysozyme(Lys),myoglobin(Mb)and catalase(Cat)with luminol respectively were first studied by chemiluminescence(CL)using flow injection(FI)technique based on the studied proteins can enhance the CL intensity of luminol.AFI-CL model of protein-luminol interaction,Ig[(I0-I)/I]= 1/nlg[P]+1/nlgKa + 2lgn,was constructed,and the interaction parameters of BSA,Lys,Mb and Cat with luminol were determined accordingly.The binding constants Ka are in the descending order of Cat>Mb>Lys>BSA at the level of 105 to 107 L mol-1,and the number of binding sites n of luminol to BSA or Lys is around 2 and to Mb or Cat is around 1.The results of thermodynamic parameters(△H,△S and △G)showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force.This work has been published in ISRNAnalytical Chemistry,2013,2013,1-5.Chapter 3 The interaction of lysozyme with lanthanide ionsThe interaction behavior of lysozyme with trivalent lanthanide ions(LnⅢ)was investigated by flow injection chemiluminescence(FI-CL)analysis.It was found that lysozyme with LnⅢ could form 1:1 association complex which remarkably quench the CL intensity from luminol-lysozyme reaction,and the quantitative correlation equation of CL intensity decrements versus the logarithm of the concentration of LnⅢ,△I =AlnCLn + B was established.The results showed the sensitive factor A varied with atomic number Z increased monotonically,and ALL of light lanthanides(LL)from LaⅢ to EuⅢ less than AHL of heavy lanthanides(HL)from GdⅢ to LuⅢ.The relationships of A vs.Z plot for LL and HL with GdⅢ break at f7 were clearly presented,and the correlations of A with physical parameters(γ±,△Hhyd and E0)of LnⅢ,were discussed.By homemade FI-CL model,lg[(I0-I)/I]= nlg[Ln]+lgK,the binding constants(K = 105-106 L mol-1 level)and binding sites(n = 1.0)were obtained,and the binding affinity abilities increased with increasing Z along the LnⅢ series.The thermodynamic parameters results showed that the reaction of lysozyme with LnⅢ was a spontaneous process by electrostatic force;the possible mechanism and application of luminol-lysozyme-LnⅢ system were given.This work has been published in RSC Advance,2014,4(36),18694-18701.Chapter 4 The interaction of myoglobin with dibutyl phthalateA sensitive method for the determination of picogram level dibutyl phthalate(DBP)in wine by flow injection chemiluminescence(FI-CL)analysis is presented for the first time,which was based on the quenching effect of DBP on the luminol-Myoglobin(Mb)CL system.The decrement of CL intensity was linearly proportional to the logarithm of DBP concentration in the range of 0.1-100.0 pg mL-1 with the detection limit of 0.03 pg mL-1(3σ).At a flow rate of 2.0 mL min-1,a complete determination of DBP including sampling and washing,could be accomplished in 0.5 min,giving the maximum sample throughput of 120 h-1.The proposed method was successfully applied to the determination of DBP in wine,human serum and urine samples with the relative standard deviations(RSDs)of less than 3.0%(n = 5).The molecule docking results showed that DBP interacted with the amino acid residues near the heme moiety of Mb.The possible CL mechanism of luminol-Mb-DBP reaction should be that the binding of Mb with DBP forming a 1:1 complex(binding constant K= 1.55 μ 104 L mol-1)led to the conformational change of Mb and resulted in the quenching of CL intensity.This work has been published in Journal of Food Composition and Analysis,2013,31,226-231.Chapter 5 The determination of LaⅢ in luminol-lysozyme systemA sensitive and fast method for the determination of LaⅢ by FI-CL analysis is presented,which was based on LaⅢ chould quench the CL intensity of luminol-lysozyme system.The decrement of CL intensity was linearly proportional to the logarithm of LaⅢ concentration in the range of 0.1-50.0 nmol L-1 with the detection limit of 0.03 nmol L-1(3σ).The proposed method was successfully applied to the determination of LaⅢ in La complexes samples with RSDs less than 2.0%(n = 7).