【作者】 田伦富；

【导师】 刘绍璞；

【作者基本信息】 西南大学 ， 分析化学， 2011， 硕士

【摘要】 本文主要研究蛋白质与[Hg(SCN)4]2-、[Zn(SCN)4]2-等金属配阴离子、蛋白质与蛋白质、蛋白质与肝素钠之间发生相互作用时对吸收光谱、荧光光谱和共振瑞利散射光谱的影响。研究探讨了它们之间相互作用的机理、结合模式、结合位点以及结合作用力等,并发展和建立了测定蛋白质和肝素钠的新方法。主要研究内容如下：1. [Hg(SCN)4]2-与蛋白质相互作用的共振瑞利散射光谱及其分析应用在pH 1.0-4.5的稀硫酸介质中,[Hg(SCN)4]2-配阴离子与人血清白蛋白(HSA)、α-糜蛋白酶(α-Chy)、牛血清白蛋白(BSA)、溶菌酶(Lyso)和Y-球蛋白(γ-G)等蛋白质反应形成复合物,此时将导致共振瑞利散射(RRS)的显著增强,也能引起吸收光谱的变化和荧光猝灭,同时还观察到圆二色(CD)光谱特征的改变。本文主要研究[Hg(SCN)4]2--蛋白质复合物的RRS光谱特征,适宜的反应条件和影响因素以及某些分析化学性质,并以[Hg(SCN)4]2--Lyso体系为例,结合吸收、荧光和圆二色光谱的变化,对复合物的结合位点、结合模式以及散射增强的原因进行了讨论。RRS法具有较高的灵敏度,它对不同蛋白质的检出限在4.6-10.8ng/mL之间,据此建立了以[Hg(SCN)4]2-配阴离子作探针测定人血液和尿液中蛋白质的新方法。2. [Hg(SCN)4]2-与血红蛋白相互作用的荧光和共振瑞利散射光谱研究在pH7.4的生理条件下,[Hg(SCN)4]2-配阴离子能与血红蛋白(Hb)发生相互作用并形成10：1的复合物,此时将导致溶液的共振瑞利散射(RRS)的显著增强并出现相应的散射光谱,其最大散射波长位于313 nm附近。本文根据反应产物对共振光散射光谱的影响,结合[Hg(SCN)4]2-对Hb的荧光猝灭作用和圆二色光谱的变化,讨论了[Hg(SCN)4]2-与Hb的相互作用、结合位点和结合模式。3. [Hg(SCN)4]2-对蛋白质的荧光猝灭反应及其分析应用在pH 1.4-3.4的酸性介质中,[Hg(SCN)4]2-配阴离子可与牛血清白蛋白(BSA)、γ-球蛋白(γ-G)和血红蛋白(Hb)等蛋白质反应形成复合物,此时将引起蛋白质荧光的猝灭。荧光猝灭程度在一定范围内与Hg(Ⅱ)的浓度成线性关系,对Hg(Ⅱ)测定的线性范围为0.015-1.25μg/mL,检出限(3σ)分别为4.4 ng/mL (BSA)、6.5 ng/mL (y-G)和12.9 ng/mL (Hb)。文中研究了适宜的反应条件和影响因素,考察了共存物质的影响,表明方法有良好的选择性,可用于红药水和乙肝疫苗中汞的测定。此外,通过研究[Hg(SCN)4]2--BSA体系吸收光谱的变化、温度的影响以及Stern-Volmer作图,判断该反应为静态猝灭反应。4. [Zn(SCN)4]2-与蛋白质相互作用的共振瑞利散射光谱及其分析应用在pH 1.75-1.90的酸性介质中,[Zn(SCN)4]2-与某些非酶蛋白质[如人血清白蛋白(HSA)、牛血清白蛋白(BSA)、血红蛋白(Hb)、γ-球蛋白(γ-G)]和某些蛋白质酶[如α-糜蛋白酶(α-Chy)、溶菌酶(Lyso)]相互作用时,不能使吸收光谱和荧光光谱发生改变,但是却能引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射(RNLS)的显著增强。不同蛋白质体系具有相似的散射光谱特征,其最大RRS、SOS和FDS波长分别位于290 nm、552 nm和392 nm附近,但相对强度有一定差异,其中以HSA、y-G和Hb体系灵敏度更高。当用RRS法时,对于HSA、y-G和Hb的检出限在4.9-7.8ng/mL之间,本文研究了反应体系的RRS光谱特征,适宜的反应条件和影响因素,考察了方法的选择性,并建立了以[Zn(SCN)4]2-作探针RRS法测定人血清和人尿液中总蛋白质的方法。文中还以[Zn(SCN)4]2--HSA体系为例讨论了复合物的结合比、它们之间的主要作用力和可能的结合模式。表明RRS法是研究蛋白质中非芳香族残基与其它分子相互作用的有效手段,这也是吸收光谱法和荧光法所不具备的优点。同时还可以从研究的体系中发展出用RRS技术高灵敏度测定蛋白质的新方法。5.胃蛋白酶与溶菌酶相互作用的共振瑞利散射和共振非线性散射光谱及其分析应用研究在pH 4.8-5.6的酸性介质中,带多个负电荷的胃蛋白酶(Pep)与带大量正电荷的溶菌酶(Lyso)可借静电引力、氢键作用力和疏水作用力而形成1：2的结合产物,这将引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射(RNLS)的显著增强,其最大RRS波长位于290 nm附近。散射增强(ΔI)在一定范围内与Lyso的浓度成线性关系,可用于痕量Lyso的测定。三种方法对Lyso的检出限分别为4.3 ng/mL(RRS)、4.5 ng/mL(SOS)和23.6ng/mL(FDS),灵敏度均较高,据此建立了以Pep作探针测定人唾液和鸡蛋清中Lyso的新方法。本文研究了反应体系的RRS、SOS和FDS光谱特征,适宜的反应条件和影响因素。干扰实验表明其他常见蛋白质基本不干扰Lyso的测定,因此本法对Lyso的测定具有较好的选择性。文中还对复合物的结合模式和散射增强的原因进行了讨论。6.肝素钠与蛋白质相互作用的共振瑞利散射和共振非线性散射光谱及其分析应用研究在适当酸度的NaAc-HCl缓冲溶液介质中,肝素钠(Hep)因其硫酸酯基离解而以带多个负电荷的大阴离子存在,而人血清白蛋白(HSA)、牛血清白蛋白(BSA)、Hb血红蛋白(Hb)、木瓜蛋白酶(Pap)和鸡卵白蛋白(Alb-egg)等蛋白质因处于其等电点(pI)之下,则以带多个正电荷的大阳离子存在,两者可借静电引力、氢键作用、疏水作用而结合形成复合物,此时将引起RRS和SOS、FDS等RNLS的显著增强并出现新的散射光谱。3种散射的增强程度(ΔI)均在一定范围内与Hep的浓度成正比,均可用于对Hep的测定,其中Hep-HSA体系的RRS法灵敏度最高,对Hep的检出限为3.1ng/mL。本文研究了适宜的反应条件,考察了共存物质的影响,表明方法有较好的选择性。基于此建立了一种灵敏度高、简便、快速测定Hep的新方法。7.肝素钠与溶菌酶相互作用的吸收、荧光和共振瑞利散射光谱研究在pH 7.4的生理条件下,带多个正电荷的碱性蛋白Lyso可通过静电引力和疏水作用力与带多个负电荷的Hep结合形成1：1的复合物,此时不仅会引起吸收光谱的变化和Lyso的荧光猝灭,而且将导致RRS显著增强,我们研究了Hep-Lyso复合物的RRS光谱特征,适宜的反应条件和影响因素以及某些分析化学性质,并结合吸收、荧光和圆二色光谱的变化,讨论了Hep与Lyso之间的反应机理和结合模式,认为静电引力和疏水作用是二者间的主要作用力,而分子体积增大、疏水界面的形成及荧光-散射共振能量转移作用是共振光散射增强的重要因素。更多还原

【Abstract】 The interaction of proteins with [Hg(SCN)4]2- and [Zn(SCN)4]2-, interaction between protein and protein, interaction between protein and sodium heparin were investigated by absorption spectra, fluorescence spectra and resonance Rayleigh scattering (RRS) spectra. The reaction mechanism, binding mode, binding sites and the main interaction forces were studied through the changes in spectra. Additional, some new methods for the determination of protein or sodium heparin were established from the investigated systems. The main work of this thesis is as follows:1. Study of the interaction between [Hg(SCN)4]2- and proteins by resonance Rayleigh scattering spectra and their analytical applications[Hg(SCN)4]2- in H2SO4 at pH 1.0-4.5 was reacted with proteins, including human serum albumin (HSA), a-chymotrypsin (a-Chy), bovine serum albumin (BSA), lysozyme (Lyso) and y-globin (y-G), to form complexes. As a result, the intensities of resonance Rayleigh scattering (RRS) were significantly enhanced. Changes in absorption, circular dichroism (CD) spectra and the fluorescence quenching of proteins were also observed. Attention was paid mainly to the RRS spectral characteristics of these complexes, suitable reaction conditions, influencing factors and a number of analytical properties. The changes in absorption, fluorescence and CD spectra for the [Hg(SCN)4]2--Lyso system were used as an example to explore the binding sites, binding model and reasons for enhanced scattering. The RRS method had a high sensitivity and the detection limits (3σ) were 4.6-10.8 ng/mL for the proteins studied. A new method is thus established herein, using [Hg(SCN)4]2- as a probe for the determination of proteins in human serum and urine samples.2. Resonance Rayleigh scattering spectral investigation of the interaction between [Hg(SCN)4]2- and hemoglobin In a slightly acidic medium of pH 1.0-4.5, anionic complex [Hg(SCN)4]2- reacted with hemoglobin to form a 10:1 complex. As a result, the intensity of resonance Rayleigh scattering and resonance nonlinear scattering such as second-order scattering and frequency doubling scattering were greatly enhanced. The discussion on the interaction, binding sites and binding model, which was based on the effect of the complexation on the resonance light scattering spectra, on the fluorescence quenching of hemoglobin by [Hg(SCN)4]2- and on the changes of absorption and circular dichroism spectroscopy, indicated that four [Hg(SCN)4]2- entered respectively the four subunits of hemoglobin to form complex with appropriate amino acid residues via hydrogen bonding, hydrophobic interaction and electrostatic forces. The enhancement of scattering was mainly due to the increase of hydrophobicity and volume of the scattering molecule, the energy transfer between fluorescence and resonance scattering, even due to the favorable conformation alteration of hemoglobin induced by [Hg(SCN)4]2-. Therefore, resonance Rayleigh scattering and resonance nonlinear scattering spectra not only can be used as a helpful tool for studying the interaction between metal complex and protein such as hemoglobin, but also can create new conditions for highly sensitive determination of trace hemoglobin.3. [Hg(SCN)4]2- quenching behavior of proteins fluorescence and its analytical application[Hg(SCN)4]2- in acidic medium of pH 1.4-3.4 was reacted with proteins, including bovine serum albumin (BSA),γ-globin (γ-G) and Hemoglobin (Hb), to form complexes. As a result, the fluorescence intensities of proteins were significantly quenched. The quenched fluorescence intensities (△F) in a certain range were linearly related to the concentration of Hg(Ⅱ), and the detection limit (3σ) were 4.4 ng/mL (BSA as a probe),6.5 ng/mL (γ-G as a probe) and 12.9 ng/mL (Hb as a probe), respectively. The effect of the interaction on the fluorescence spectral characteristics of these complexes, suitable reaction conditions and influencing factors were investigated. The quenching mechanism was studied by absorption spectroscopy, binding constant and corresponding thermodynamic parameters at different temperatures. Some potential interferents on the assay by BSA were investigated. The method displayed good selectivity and was satisfactorily applied to the determination of asceptichrome in mercurochrome solution and thiomersalatum in HB-vaccine.4. Study on the interaction between [Zn(SCN)4]2- and proteins by resonance Rayleigh scattering spectra and their analytical applications In pH 1.75-1.90 acid medium, the interactions of [Zn(SCN)4]2- with proteins, such as human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), a-chymotrypsin (a-Chy), lysozyme (Lyso) and y-globin (y-G), could not induce the change in absorption spectra and fluorescence spectra, but these interaction could result in the great enhancement of resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS). The maximum scattering wavelength was located at 290 nm (RRS),552 (SOS) nm and 392 nm (FDS), respectively. These six systems have different sensitivity, the detection limit (3σ) of HSA、y-G and Hb by RRS method is 4.9-7.8 ng/mL which is the highest sensitivity. The RRS spectral characteristics of these systems, suitable reaction conditions, influencing factors and effect of coexisting substances were investigated. Thus a new method for the determination of total proteins in human serum and urine was established. Additional, the binding stoichiometric ratio, the main interaction forces and possible binding mode were studied when [Zn(SCN)4]2- reacted with HSA.5. Study on the interaction between pepsin and lysozyme by resonance Rayleigh scattering and resonance non-linear scattering and its analytical applicationIn pH 4.8-5.6 acid medium, multiple negatively charged pepsin (Pep) could bind to multiple positively charged lysozyme (Lyso) to form 1:2 complex via electrostatic attraction, hydrophobic interaction and hydrogen bonding. As a result, the intensities of resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS), including second order scattering (SOS) and frequency doubling scattering (FDS), could be greatly enhanced. The maximum RRS wavelength was at about 290 nm where the enhanced intensity (△I) was linear to the concentration of Lyso in a certain range. The detection limit (3σ) of Lyso was 4.3 ng/mL (RRS),4.5 ng/mL (SOS) and 23.6 ng/mL(FDS). The RRS method was utilized to determine the level of Lyso in human saliva and hen egg white.6. Study on the interaction between sodium heparin and proteins by resonance Rayleigh scattering and resonance non-linear scattering and its analytical applicationIn NaAc-HCl buffer medium with proper pH, some positively charged proteins such as human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), Papain (Pap) and albumin egg (Alb-egg) could react with negatively charged sodium heparin (Hep) to form complexes by electrostatic attraction, hydrophobic interaction and hydrogen bonding. As a result, the intensities of resonance Rayleigh scattering (RRS), second order scattering (SOS) and frequency doubling scattering (FDS), could be significantly enhanced. The enhanced intensity (△I) was linear to the concentration of Hep in a certain range. The detection limit (3σ) of Hep by RRS was 3.1 ng/mL. The effects of coexisting substances showed good selectivity of the established method.7. Study on the interaction between sodium heparin and lysozyme by absorption, fluorescence and resonance Rayleigh scattering spectraUnder pH 7.4 physical condition, multiple positively charged lysozyme (Lyso) could bind to multiple negatively charged sodium heparin (Hep) to form 1:1 complex via electrostatic attraction and hydrophobic interaction. As a result, the change in absorption spectra, the fluorescence quenching of Lyso and the great enhancement of resonance Rayleigh scattering (RRS) intensity were observed. The RRS spectral characteristics, suitable reaction conditions, influencing factors and some analytical properties were investigated. The reaction mechanism and binding mode were studied through the changes in absorption, fluorescence, RRS and circular dichroism spectra.更多还原