摘要
作为组成生命最为重要的两类物质,核酸和蛋白质对一切生命活动都有至关重要的作用。核酸是遗传的物质基础,它的结构直接影响其功能;而蛋白质是生物体含量最丰富的高分子物质,在生物体内有重要的功能和地位。
植物生长调节剂的使用促进了现代农业生产的发展,然而随着植物生长调节剂品种和用量的不断增加,它在食品和环境中的残留所引发的一系列问题越来越突出,对人类健康与环境的危害已引起广泛的关注。同时,对植物生长调节剂残留物及其代谢物的高灵敏度检测也变得尤为重要。
本论文利用荧光技术、共振光散射技术、吸收光谱技术、圆二色谱技术、透射电镜等手段研究了植物生长调节剂与生物大分子的作用机理,建立了植物生长调节剂快速、准确、简便的分析方法。论文共分五个部分。
论文的第一部分主要综述了药物与生物大分子相互作用的研究方法和研究进展,以及共振光散射技术基本原理及其分析应用的研究进展等。共引用文献193篇。
论文的第二部分中,利用荧光光谱法、紫外光谱法、圆二色光谱法等方法研究了6-苄氨基嘌呤(6-Benzylaminopurine,缩写为6-BA)与牛血清白蛋白(BSA)之间的相互作用机理。结果表明:6-BA可以显著猝灭BSA的荧光,其猝灭机制为静态猝灭。6-BA与BSA的结合常数在293K、298K和308K时分别为2.4×10~4、2.6×10~4和3.1×10~4 L/mol,两者之间存在一个结合位点。体系的焓变△H和熵变△S分别为14.2 KJ/mol和132.1 J/(mol·K),说明6-BA与BSA之间主要以疏水作用力相互结合。根据F(?)rster非辐射能量转移理论,6-BA与BSA间的结合距离为2.99nm,能量转移效率为0.24。紫外光谱和圆二色光谱实验结果表明,6-BA可以导致BSA构象发生改变。
论文的第三部分中,利用荧光光谱法、紫外光谱法、荧光寿命和圆二色光谱法等方法研究了6-糠氨基嘌呤(Kinetin,缩写为KT)与牛血清白蛋白之间的相互作用机理。结果表明:KT可以显著猝灭BSA的内源荧光,其猝灭机制为静态猝灭,其猝灭常数K_(SV)在288K和303K时分别为1.45×10~4和1.42×10~4L/mol,Stern-Volmer曲线是两段相交于C_(KT)=8.0×10~(-5)mol/L的回归曲线,说明KT与BSA之间存在两类结合位点。数据处理及热力学参数计算表明:低浓度KT-BSA(C_(KT)＜8.0×10~(-5)mol/L)的结合主要是疏水作用,结合位点数约为1;较高浓度KT(C_(KT)＞8.0×10~(-5)mol/L)主要以氢键、范德华力与BSA结合,结合位点数3.7。根据F(?)rster非辐射能量转移理论计算了KT与BSA间的结合距离,紫外光谱和圆二色光谱实验结果表明,KT的存在使BSA二级结构发生了改变。
论文的第四部分中,利用荧光光谱法研究了α-萘乙酸(1-naphthlcetic acid,缩写为NAA)与DNA之间的相互作用。光谱实验结果表明,DNA能使NAA的荧光强度猝灭,根据荧光强度变化和荧光猝灭公式求得NAA与DNA的荧光猝灭常数,荧光寿命实验结果表明其荧光猝灭是由形成复合物的静态猝灭过程所致。黏度实验、热变性实验、离子强度等多种实验结果表明,NAA与DNA间的结合作用为部分插入式和沟槽式相结合。
论文的第五部分中,研究了在pH为4.5的HAc-NaAc缓冲溶液中,6-BA对纳米银-SDS体系的共振光散射的增强效应。研究发现,6-BA的加入可以使nanoAg-SDS体系的共振光散射强度大大增强,由此建立了一种测定6-BA的简单、灵敏的共振光散射法。在最佳实验条件下,增强的共振光散射强度与6-BA浓度之间呈线性关系。在nanoAg-SDS-6-BA体系中,6-BA的线性范围为2.0×10~(-8)-1.0×10~(-5)mol╱L,检出限为2.8×10~(-9)mol/L,该法已应用于合成样品中6-BA的定量测定,得到满意的结果。同时通过透射电子显微技术、电导率测定技术,电势电泳技术等探讨了体系共振光散射增强的机理。
本论文的主要特点是:
(1)本文利用荧光光谱、紫外吸收光谱、荧光寿命和圆二色光谱等多种手段讨论了植物生长调节剂6-苄氨基嘌呤和6-糠氨基嘌呤与牛血清白蛋白之间的相互作用,丰富了农药与蛋白质相互作用的研究内容。
(2)本文利用荧光光谱法、紫外吸收光谱法与黏度法等多种实验方法研究了NAA与DNA之间的相互作用。
(3)研究了在pH为4.5的HAc-NaAc缓冲溶液中,6-BA对纳米银-SDS体系的共振光散射的增强效应。建立了灵敏检测6-BA的新方法。该方法简便、快速,具有较宽的线性范围。并利用荧光光谱、紫外吸收光谱、透射电子显微镜等多种实验手段对体系的作用机理进行了研究。
As the material base of life,nucleic acids and proteins play an important role in the action of life.There are connection between nucleic acids configuration and its function.Proteins are most abundant in organism,and they attach themselves almost to all life action.
The application of plant growth regulators promoted the development of modern agriculture.However,along with the continuous increase of variety and quantity of the plant growth regulators,the serial food safety and environment pollution problems coursed by the plant growth regulators residua become more and more serious,the harm to the human health and the environment courses widely international attention. At the same time,high-sensitivity detection method becomes very important to the plant growth regulators residua and their metabolites.
For the seek of researching the interaction of bio-macromolecules and plant growth regulators,this thesis studied the interaction mechanism between bio-macromolecules and plant growth regulators using the research techniques including fluorescence,Resonance Light Scattering(RLS),UV absorption,circular dichroism(CD)spectrometries and transmission electron microscopy(TEM).Some rapid,accurate and handy assays were developed for plant growth regulators.This thesis was divided into five sections.
In the first section,we summarized the research method and evolution of the interaction between the drugs and bio-macromolecules,the principle of RLS method and the recent developments of RLS.193 references were cited here.
In the second section,the interaction between 6-Benzylaminopurine(6-BA)and bovine serum albumin(BSA)was investigated by fluorescence,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 6-BA with BSA at 293K,298K and 308K were obtained as 2.4×10~4,2.6×10~4 and 3.1×10~4 L/mol,respectively.There was one single binding site between 6-BA and BSA.The thermodynamic parameters enthalpy change (ΔH)and entropy change(ΔS)were calculated as 14.2 kJ/mol and 132.1 J/(mol·K), respectively,which indicated that the acting force between 6-BA and BSA were mainly hydrophobic interactions.According to the F(?)rster non-radiation energy transfer theory,the average binding distance between donor(BSA)and acceptor (6-BA)was obtained(r=2.99nm).The investigations of the UV/Vis and CD spectra of the system showed that the conformation of BSA was changed in presence of 6-BA.
In the third section,the interaction mechanism of Kinetin(KT)with BSA was studied by the methods of fluorescence,fluorescence lifetime,UV absorption and CD spectrometries.KT could strongly quench the intrinsic fluorescence of BSA by static quenching.The binding constants of KT with BSA at two temperatures(288K and 303K)were obtained as 1.45×10~4 and 1.42×10~4 L/mol.The Stern-Volmer curve had an intersection at C_(KT)=8.0×10~(-5)mol/L,which indicated that KT bound to different binding sites on BSA.The analytical results of fluorescence data showed when C_(KT)＜8.0×10~(-5)mol/L,the number of binding sites was near 1,and when C_(KT)＞8.0×10~(-5) mol/L,the number of binding sites was approximately 3.7.The thermodynamic parameters were calculated by Van't Hoff equation.The enthalpy change enthalpy change(ΔH)and entropy change(ΔS)were calculated at two concentration of KT. The results suggested that the hydrophobic interaction might play a main role in the interaction of KT(C_(KT)＜8.0×10~(-5)mol/L)with the BSA,while C_(KT)＞8.0×10~(-5)mol/L the hydrogen bonding and Vander Waals forces became major.The binding distance(r) between KT and tryptophan in BSA was obtained according to the F(?)rster energy transfer theory.The investigations of the UV/Vis and CD spectra of the system showed that the secondary structure of BSA was changed in presence of KT.
In the fourth section,the binding characteristics and mechanism of DNA to 1-naphthlcetic acid(NAA)had been investigated by using the methods of fluorescence spectroscopy and viscosity.The spectroscopy analysis showed that DNA could quench the endogenous fluorescence of NAA.Based on the changes of fluorescence intensity and fluorescence quench formulas,the quenching constants between DNA and NAA had been worked out,and the fluorescence lifetime results showed that the quenching effect of DNA on NAA was a static fluorescence mechanism.The experimental results of viscosity,thermal denaturation,ionic strength showed that the binding mode of NAA with DNA was partial intercalation and groove binding.
In the fifth section,the RLS enhancing effect of 6-BA-nanoAg-SDS was studied in HAc-NaAc(pH=4.5)buffer.It was found that 6-BA could enhance the RLS intensity of nanoAg-SDS system.Under optimum conditions,the calibration graph for 6-BA was obtained,the enhanced intensity of RLS at 350 nm was in proportion to the concentration of 6-BA in the range of 2.0×10~(-8)-1.0×10~(-5)mol/L.The detection limit (S/N=3)is 2.8×10~(-9)mol/L,and the results for the determination of 6-BA in synthetic samples was satisfactory.And the interaction mechanism of 6-BA,SDS and nanoAg was studied by several techniques including TEM,measurement of conductivity,Zeta potentiometry and so on.
The chief characteristics of this thesis were as follows:
(1)In this thesis,we studied the interaction mechanisms between BSA and plant growth regulators using fluorescence,fluorescence lifetime,UV absorption and CD spectrometries,which enriched the research in the fields of pesticide.
(2)In this thesis,we used variety methods of fluorescence spectroscopy, absorption spectroscopy and viscosity to investigate the binding characteristics and mechanism between DNA and NAA.
(3)The RLS enhancement of system of 6-BA-nanoAg-SDS was studied,which was utilized to determine of trace amount 6-BA.The method was simple,rapid and also had a wide concentration linear range.Several instruments were used to study the interaction mechanism of the system such as fluorescence,UV absorption and TEM.
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