| Proteins and nucleic acids(nucleoside) are the material base of life,which take on diversified physiological function.Proteins are the carrier of many physiological functions,and are also the direct expresser of physiological characters.Nucleic acids (nucleoside) are related to heredity,tumor,the effect of virus and so on.So it is of special value to study the interaction of small molecule with biomolecules and to develop the quantitative analysis of biomolecule with high sensitivity and low detection limit.This project is the forward position and hot point in biochemical and biophysical researches.This thesis studies the interaction mechanism between biomolecules and small molecules using the techniques including fluorescence,UV-visible absorption, circular dichroism(CD),resonance light scattering(RLS) and Fluorescence lifetime. Some rapid,accurate and handy assays are developed for nucleoside and nucleic acids The main conclusions are listed as follows:In the first section,we summarized the structure and character of protein,the research method and evolution of protein with small molecules,and recent developments of fluorescence probe for both nucleic acids and nucleoside.In the second section,the interaction between pymetrozine(Py) and bovine serum albumin(BSA) were studied in physiological condition using fluorescence,UV absorption and CD spectrometries.Py could strongly quench the intrinsic fluorescence of BSA by static quenching.The binding constructs of Py with BSA at two temperatures(289 K and 299 K) are 5.4×104 and 2.7×104 L/mol.There was a single binding site between Py and BSA.The thermodynamic parameters were calculated by Van't Hoff equation,the enthalpy change(ΔH) and entropy change(ΔS) are -41.8 KJ/mol and -81.7 J/(mol·K),respectively.The results suggested that the acting force between Py and BSA was mainly hydrogen bond and Vander Waals force. The average binding distance between donor(BSA) and acceptor(Py) was obtained(r =2.4nm).The investigations of the UV/Vis and CD spectra of the system showed that the secondary structure of BSA was changed in presence of Py.In the third section,the interaction between flumetsulam(FLU, N-(2,6-difluorophenyl)-5-methyl(1,2,4)-triazolo(1,5-a)pyrimidine-2-sulfonamide) and bovine serum albumin(BSA) was investigated by fluorescence,fluorescence lifetime, UV absorption and CD spectrometries.A strong fluorescence quenching was observed and the quenching mechanism was considered as static quenching according to the Stern-Volmer equation.The binding constants of FLU with BSA at 299K and 309K were obtained as 3.9×104L/mol and 3.2×104 L/mol,respectively.There was a single binding site between FLU and BSA.The thermodynamic parameters enthalpy change (ΔH) and entropy change(ΔS) were calculated as -15.19 KJ/mol and 37.06 J/(mol·K), respectively,which indicated that the electrostatic interaction force played major roles in the binding reaction of FLU with BSA.According to the F(o|¨)rster non-radiation energy transfer theory,the average binding distance between donor(BSA) and acceptor(FLU) was obtained(r=3.6 nm).The investigations of the UV/Vis and CD spectra of the system showed that the conformation of BSA was changed in presence of FLU.In the forth section,it was found that guanine could enhance the fluorescence of quercetin.Based on this,a new method for the determination of guanine was proposed.In BR buffer solution(pH=5.60),the fluorescence intensity of quercetin system could be greatly enhanced by guanine and the enhanced intensity was in proportion to the concentration of guanine in the range 1.0×10-9～9.0×10-6mol/L, its detection limits was 2.0×10-10 mol/L.Interference test showed that the other three bases in nucleic acid had little effects on the determination of guanine.And the mechanism study indicated that the strong interaction and the form of 1:1 complex between guanine and quercetin resulted in the depolymerization of the quercetin aggregation and fluorescence enhancement of this system.Actual samples were determined satisfactorily.In the fifth section,we found that there were interactions among HP-β-CD,ANS and DNA.It was found that the weak fluorescence of ANS was enhanced greatly by the addition of HP-β-CD,and DNA could quench the fluorescence of the system.In BR buffer solution(pH=4.20),the decreased fluorescence intensity was in proportion to the concentration of nucleic acid in the range 2.5×10-8-3.0×10-5 g/ml for fsDNA, 5.0×10-9-4.0×10-5 g/ml for ctDNA,2.5×10-8-5.0×10-5 g/ml for yRNA.Their detection limits were 8.0×10-9,3.6×10-9 and 6.6×10-9 g/ml(S/N=3),respectively. Samples were determined satisfactorily.The interaction mechanism investigation indicated that HP-β-CD could cause the change of double helix structure in nucleic acid,and therefore HP-β-CD-ANS complex interacted with nucleic acid easily, which resulted in the fluorescence intensity decreased.The chief characteristics of this thesis are as follows:(1) In this thesis,we studied the interaction mechanisms between BSA and small moleculars such as a new herbicide flumetsulam and a new pesticide pymetrozine using fluorescence,fluorescence lifetime,UV absorption and CD spectrometries, which enriched the research in the fields of herbicide and pesticide.(2) It is found that guanine can enhance the fluorescence of quercetin.Based on this, a new method for the determination of guanine is proposed.The method is simple, rapid,sensitive and selective.(3) It is found that DNA could quench the fluorescence of the HP-β-CD-ANS system in BR buffer solution(pH=4.20),and the decreased fluorescence intensity was in proportion to the concentration of nucleic acid.Based on this,a new method for the determination of nucleic acid is proposed.This method is simple,sensitive and quick.
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