Integrated Spectroscopy Analytical Technique For Investigation Of Small Molecules Interacting With Proteins

The interactions between small molecules and biological molecules are importantfor chemistry biology investigation.Serum albumin,the most abundant carrier proteinin blood plasma,can bind many exogenous and endogenous compounds extensively.IgG (gammaglobulin) is one of the most impartment immunoglobulin in organism.Itis most abundant in normal human serum.IgG is capable of binding with drugs,smallorganic molecules and related antigen reversibly.Investigation on the bindingmechanism of small molecules with serum albumin and immunoglobulin is muchimportant in pharmacokinetics and toxicology.Thus,it became an interesting researchfield of life sciences,chemistry and clinical medicine.In this dissertation,on the basisof the previous research,the following major innovative works were carried out:(1) The molecular modeling method was used to study the binding mode of dyemolecule,active components of Chinese medicines with human gammaglobulin(HGG) and human serum albumin (HSA),obtain the molecule model of smallmolecules with proteins,giving the referenced information for the followingexperiment.(2) Integrated spectroscopy analytical technique including fluorescence spectrum,uv absorption spectrum,fourier transform infrared (FT-IR) spectrum and circulardichroism (CD) spectrum were used to study the interactions of dye molecule,activecomponents of Chinese medicines with HGG and HSA,and the results obtained fromevery method were in good agreement.This also verified the results of molecularmodeling.(3) The binding constants,the numbers of binding site and the interaction forceinvolving in the interaction of small molecules and proteins were confirmed byScatchard equation,Sips equation and Van't Hoff equation.(4) The secondary structure compositions of HSA and their small moleculescomplexes were estimated by quantitative analysis using IR self deconvolution withsecond-derivative resolution enhancement and curve-fitting procedure.(5) The binding locations of small molecules on HSA were confirmed by competitive binding experiments,the results verified the results of molecularmodeling technique.(6) The information obtained from the synchronous fluorescence spectra wereused to determine the microenvironment changes of chromophore in different smallmolecule-protein systems.(7) The interactions between active components of Chinese medicines and HSAwere studied in AOT/isooctane/water microemulsions,which open up the new studyfield for the interaction between proteins and small molecules.This dissertation consists of four chapters.Chapter 1:The structures,functions of proteins were briefly introduced firstly.The developments of interaction of small molecules with proteins were reviewed,andthe methods used to study the interaction of small molecules with proteins weresummarized.Chapter 2:The interaction of HGG with small dye molecule neutral red wasstudied by integrated spectroscopy analytical technique combined with molecularmodeling study.The binding mode between neutral red and HGG were studied bymolecular docking technique,and the molecular model between them was established.The binding parameters including the average affinity constant,the number of bindingsites and the thermodynamic parameters of this dye bound to HGG were calculatedaccording to Sips method and Van't Hoff equation,respectively.And the main actingforces between neutral red and HGG were discussed.The effects of this molecule onthe secondary structure of HGG were investigated by FT-IR and CD techniques.Chapter 3:The interactions between paeonol,naringin and HSA,HGG wereinvestigated by spectroscopy methods and the molecular modeling technique,respectively.The binding regions and binding modes of the two drugs on proteinswere obtained from the molecular modeling results.To compare the results,it showedthat the two drugs could bind with proteins and quench the fluorescence of proteins.The fluorescence methods were used to calculate the binding constants,the number ofbinding sites and thermodynamic parameters of the reactions.The results from FT-IRand CD spectra indicated the different degree on the changes of the secondary structure compositions of proteins and their drugs complexes.Chapter 4:The interactions between bergenin,esculin,gallic acid and caffeic acidwith HSA in AOT/isooctane/water microemulsions were characterized for the firsttime using fluorescence spectroscopy in combination with UV absorptionspectroscopy,FT-IR spectroscopy,CD spectroscopy and dynamic light scattering(DLS) technique.The molecular modeling method was applied to study theoreticallythe binding regions and binding modes for four drugs with HSA.The spectroscopydata suggested that a less polar environment of amino acid residues was provided bymicroemulsion and the mobility of the chromophores of HSA in microemulsion wasmore confined than in aqueous solution.The binding of HSA to drugs inmicroemulsion quenched the intrinsic fluorescence of HSA and the binding constantsdecreased with the increasing temperature.And the main acting forces between drugsand HSA were discussed through thermodynamic parameters of the reactions.FT-IRand CD data suggested that the protein conformations were altered after binding withdrugs.The DLS data suggested the possible location of HSA in microemulsion andproved the interaction between drugs and HSA.