【作者】 徐巍；

【导师】 吴霞；

【作者基本信息】 山东大学 ， 分析化学， 2009， 硕士

【摘要】 蛋白质与核（苷）酸是构成机体重要的生物分子。蛋白质肩负着各种生理上的功能,从整体上维持生物体新陈代谢活动的进行,是生物性状的直接表达者。核（苷）酸与生物的遗传变异、肿瘤的发生、病毒的感染、射线对机体的作用有着重要关系。因此研究生物分子与小分子相互作用,建立高灵敏度、低检测限的生物分子的测定方法,对分子水平上阐明生命的奥秘等方面具有重要的意义,是当前生物分析化学研究的前沿和热点之一。本论文利用荧光技术、共振光散射技术、吸收光谱技术、圆二色光谱技术等手段研究了小分子物质与生物分子的作用机理,建立了对核（苷）酸快速、准确、简便且选择性的分析方法。论文共分五个部分。论文的第一部分主要综述了蛋白质结构和性质、小分子物质与蛋白质相互作用的研究方法和研究进展,以及核（苷）酸荧光探针研究进展和发展趋势。论文的第二部分应用荧光光谱、紫外吸收光谱和圆二色光谱等方法研究了生理条件下杀虫剂吡蚜酮（Py）与牛血清白蛋白（BSA）的相互作用机理。结果表明,在生理条件下,Py对BSA有较强的荧光猝灭作用,其荧光猝灭方式以静态猝灭为主。Py通过氢键或范德华作用力与BSA相结合.它们的结合服从位点结合模型,形成具有一定稳定性的1:1的复合物。Py的加入使BSA骨架结构周围微环境的极性发生了变化,导致BSA的构象改变,α-螺旋含量升高,发生了去折叠过程。论文的第三部分利用荧光光谱法、紫外光谱法、圆二色光谱法和荧光寿命等方法研究了除草剂唑咪磺草胺（Flumetsulam,缩写为FLU）与血清白蛋白之间的相互作用机理。结果表明:FLU可以显著猝灭BSA的荧光,其猝灭机制为静态猝灭。FLU与BSA的结合常数K在299 K和309 K时分别为3.9x10⁴ L/mol和3.2x10⁴ L/mol,两者之间存在一个结合位点。体系的焓变AH和熵变AS分别为-15.19 KJ/mol和37.06 J/（mol·K）,说明FLU与BSA之间主要以静电引力相互结合。根据F（o|¨）rster非辐射能量转移理论,FLU-BSA间的结合距离为3.6 nm,能量转移效率为0.22。紫外光谱和圆二色光谱实验结果表明,FLU可以导致BSA构象发生改变。在论文的第四部分,用槲皮素作为荧光探针,建立了灵敏而选择性测定鸟嘌呤的新方法。结果表明,在BR缓冲溶液中,鸟嘌呤碱基加入到槲皮素体系中,荧光强度显著增强且增强程度与鸟嘌呤碱基的浓度有良好的线性关系。该方法可用于鸟嘌呤的测定。在最佳实验条件下,鸟嘌呤的线性范围为1.0×10^-9～9.0x10^-6mol/L,检出限为2.0x10^-10 mol/L,干扰实验表明其它碱基对鸟嘌呤的测定影响较小。机理研究认为,鸟嘌呤对槲皮素的荧光增强作用源于鸟嘌呤对槲皮素的荧光增强来源于槲皮素超分子聚合物的解聚及鸟嘌呤-槲皮素氢键缔合物的形成。该方法用于实际尿样中鸟嘌呤的测定,结果令人满意。论文的第五部分研究了HP-β-CD、ANS和核酸的相互作用。研究表明,HP-β-CD包结了ANS,使ANS的荧光强度显著增强,而核酸（fsDNA、ctDNA和yRNA）的加入,可使得体系的荧光强度降低,且降低程度大大超过ANS-核酸体系的荧光猝灭作用,体系的荧光猝灭程度与核酸的浓度在一定范围内具有良好的线性关系,据此建立了测定核酸的新方法。fsDNA、ctDNA和yRNA的线性范围分别为:2.5x10^-8-3.0×10^-5 g/ml,5.0×10^-9-4.0x10^-5 g/ml和2.5x10^-8-5.0×10^-5 g/ML。检出限分别为8.0×10^-9 g/ml,3.6×10^-9 g/ml和6.6x10^-9 g/ml。并成功应用于合成样品中fsDNA的定量测定。机理研究表明,HP-β-CD与fsDNA相互作用导致其双螺旋结构发生改变,从而使HP-β-CD-ANS更容易与核酸的碱基发生作用,导致体系荧光猝灭。本论文的主要特点:（1）本文利用荧光、荧光寿命、紫外吸收、圆二色光谱等多种实验手段研究磺酰胺类除草剂唑咪磺草胺（flumetsulam,缩写为FLU）和杀虫剂吡蚜酮（Py）与牛血清白蛋白之间的相互作用机理,丰富了农药等有机小分子与蛋白质作用的研究。（2）研究发现,在弱酸条件下鸟嘌呤能明显增强槲皮素的荧光强度,而DNA中其他三种基本碱基的干扰较小,据此建立了选择性测定鸟嘌呤的荧光光度法。本法还具有操作简便、快速、线性范围宽、灵敏度高等特点。（3）研究发现,在pH为4.20的BR缓冲溶液中,核酸的加入可使HP-β-CD-ANS体系发生荧光猝灭,且猝灭程度与核酸的浓度呈线性关系,从而建立了灵敏测定核酸的荧光新方法,该方法简便、快速,具有较宽的线性范围。更多还原

【Abstract】 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.更多还原