喹诺酮药物与血清蛋白相互作用的三维荧光光谱研究

应用三维荧光光谱和三维荧光偏振光谱研究了数种喹诺酮药物与牛血清白蛋白(BSA)分子间的相互作用。由三维荧光(偏振)光谱得到的指纹信息说明了喹诺酮药物与BSA结合反应对BSA分子构象的影响。通过研究喹诺酮药物发生相互作用前后BSA荧光偏振度及各向异性的变化,定量说明了喹诺酮药物-BSA所发生的结合反应。     

分子内电荷转移荧光探针1-酮-2-（对二甲氨基苯亚甲基）四氢萘与人血清白蛋白作用

采用荧光及紫外光谱研究了1-酮-2-（对二甲氨基苯亚甲基）-四氢萘（KDTN）与人血清白蛋白（HSA）相互作用的光谱特性。 结果表明,静态猝灭和非辐射能量转移是导致KDTN对HSA荧光猝灭的主要原因。 测得17、27和37 ℃ 3个温度下的结合常数K_A分别为1.633×10⁸、0.7998×10⁸和0.347×10⁸ L/mol,结合位点数n分别为1.7、1.6和1.7;据Forster偶极 偶极非辐射能量转移理论,计算得到KDTN与HSA在3个温度下的作用距离r分别为2.64、2.59和2.64 nm;能量转移效率E分别为0.5100、0.4797和0.4210。 热力学参数表明,二者主要以范德华力或氢键结合;用同步荧光技术研究了KDTN对HSA构象的影响,结果表明,KDTN的加入对HSA构象影响不大。     

溶液中溶菌酶的荧光光谱研究

本文采用荧光分析法(荧光光谱、荧光偏振度)结合内源荧光探针色氨酸残基对溶菌酶及其在不同条件下的构象变化进行了研究。结果表明,利用荧光光谱可对溶菌酶溶液构象进行有效的分析,能够较直观地表征色氨酸残基在溶菌酶分子中的微环境及其在不同条件下构象的变化,得出了一些较有价值的结果。     

槲皮素与酪蛋白和牛血清白蛋白的相互作用及共存碳纳米管的影响

采用荧光猝灭和同步荧光法,研究了磷酸缓冲溶液(PBS, pH=7.4)中有无碳纳米管(CNTs)共存时,荧光活性物质槲皮素(Qct)与牛血清白蛋白(BSA)和酪蛋白(Cas)的相互作用. 推导了方法1(固定蛋白质浓度, 改变Qct浓度, 测量蛋白质荧光改变)和方法2(固定Qct浓度, 改变蛋白质浓度, 测量Qct荧光改变)研究分子间作用的一般方程, 由非线性最小二乘拟合法测算了结合常数K和摩尔结合比n, 并藉此定量评估了“光内滤所致猝灭”效应的影响. 研究了共存CNTs或Qct对BSA或Cas的荧光猝灭效应, 及CNTs对Qct-BSA和Qct-Cas相互作用的影响. 以同步荧光法考察了CNTs或Qct对BSA或Cas构象的影响, 并测算了CNTs或Qct与蛋白质中酪氨酸(Tyr)或色氨酸(Trp)残基相关的K和n. 结果表明, CNTs主要与处于蛋白质分子表面附近的Trp残基作用, 而小分子Qct则还可与处于蛋白质分子内部的Tyr残基作用.     

磺胺嘧啶钠探针分析生物样品中蛋白质含量的研究

在pH 7.4的Tris-HCl缓冲溶液中,荧光猝灭光谱和三维荧光光谱显示磺胺嘧啶钠(SDS)可与人血清白蛋白(HSA)相互作用,使人血清白蛋白疏水微环境极性以及构象发生变化。考察了Δλ值、反应介质、离子强度等因素对该体系同步荧光光谱特征及强度的影响。在选定的最佳实验条件下,体系的同步荧光强度(ISF)与人血清白蛋白在1.38～276μg/mL的浓度范围内呈现良好的线性关系,线性相关系数为0.9992,从而建立了以磺胺嘧啶钠为分子探针,用固定波长同步荧光光谱分析测定蛋白质的新方法,检测限可达0.48μg/mL。用此方法对生物样品人血清、唾液和尿液中蛋白质含量进行了测定并进行加标回收实验,回收率在95.7%～103.7%之间。本文还用经典的考马斯亮蓝法对样品进行了测定,所得结果和本方法一致。同时还考察了一些常见的共存物质对蛋白质测定的影响。     

甘草酸二铵与牛血清白蛋白相互作用的光谱

在模拟人体生理条件下,用常规荧光光谱和紫外－可见吸收光谱研究甘草酸二铵和牛血清白蛋白的相互作用机制,并利用同步荧光光谱和三维荧光光谱研究了药物对牛血清白蛋白构象的影响。研究表明,甘草酸二铵对牛血清白蛋白具有荧光猝灭作用,且为单一的动态猝灭过程。根据Stern-Volmer方程求出了两者相互作用的动态猝灭常数,并根据Förster非辐射能量转移理论确定了药物与蛋白的结合距离。     

光谱法研究硫鸟嘌呤与七元瓜环及牛血清白蛋白的超分子相互作用

利用紫外-可见吸收光谱法和荧光光谱法研究了抗癌药物硫鸟嘌呤(6-TG)与七元瓜环(Q[7])及牛血清白蛋白(BSA)的相互作用. 结果表明, 6-TG与Q[7]及BSA可形成三元复合物, 且6-TG与Q[7]及BSA均可形成1:1的超分子配合物, 6-TG能引起BSA的荧光猝灭, 猝灭机制为静态猝灭. 此外, 还用同步荧光法和三维荧光法考察了6-TG对BSA构象的影响, 结果表明6-TG的加入使BSA的构象发生了变化, 而同步荧光光谱结果表明结合位点更接近于色氨酸.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法, 表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息. 同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境; 二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光, 使其构象发生变化. 荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息, 揭示KA的存在使HSA的流动性和微粘度发生变化. 定量求得不同温度下(298、308 和318 K)考拉维酸与HSA作用的键合参数和热力学参数. 分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内, 并与赖氨酸Lys195 和天冬氨酸Asp451 形成三个氢键, 与HSA的键合模式主要是疏水作用; 位点竞争实验证明考拉维酸在HSA亚结构域的位点Ⅱ位发生作用. 另外, 获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制. 结果表明, HSA对考拉维酸有较强的结合能力, 提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息.同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境;二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光,使其构象发生变化.荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息,揭示KA的存在使HSA的流动性和微粘度发生变化.定量求得不同温度下(298、308和318 K)考拉维酸与HSA作用的键合参数和热力学参数.分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内,并与赖氨酸Lys195和天冬氨酸Asp451形成三个氢键,与HSA的键合模式主要是疏水作用;位点竞争实验证明考拉维酸在HSA亚结构域的位点II位发生作用.另外,获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制.结果表明,HSA对考拉维酸有较强的结合能力,提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

The Photochemical Study of HSA and BSA with Resonance Light-Scattering and Fluorescence Spectra

In recent years, people have paid close attention to the physiological harms induced byultraviolet (UV) irradiation. The serum albumin, which constitutes 60% of blood plasma,has very important physiological functions. Therefore, to study their photochemicalreaction is of great significance. The metal ions, little molecules and medicines etcinteracting with HSA or BSA have been reported ','*"', but it has not been repoFted aboutusing RLS to study the photochemical reaction of HSA or BSA.…     

Spectroscopic Study on the Interaction between Bovine Serum Albumin and Tween‐20

The interaction between nonionic surfactant Tween-20 and bovine serum albumin (BSA) is studied in Tris‐HCl buffer solution by spectroscopic methods. The intrinsic fluorescence of BSA is quenched by the addition of Tween‐20. The UV‐visible absorption spectra and the synchronous fluorescence spectra show that the addition of Tween‐20 changes the polarity of the environment around tryptophan (trp) residues of BSA. The fraction of trp residues on the surface of BSA with and without Tween- 20 is calculated via I^? quenching experiments.     

Investigation of the association behaviors between biliverdin and bovine serum albumin by fluorescence spectroscopy

The interaction between biliverdin and bovine serum albumin (BSA) has been studied by steady fluorescence spectroscopy, synchronous fluorescence and resonance light scanning spectra. The binding of biliverdin to BSA quenches the tryptophan residue fluorescence and the results show that both static and dynamic quenching occur together with complex formation. The binding constant and binding sites of biliverdin to BSA at pH 7.1 are calculated to be 3.33 × 10⁸ L/mol and 1.54, respectively, according to the double logarithm regression curve. In addition, the distance between the biliverdin and BSA is estimated to be 1.25 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues has not obvious changes, which obeys the phase distribution model. Finally, the thermodynamic data show that biliverdin molecules enter the hydrophobic cavity of BSA via hydrophobic interaction.     

Interactions of Quercetin with Casein and Bovine Serum Albumin as well as the Effects of Coexisting Carbon Nanotubes

Fluorescence quenching and synchronous fluorescence methods were used to study the interactions of fluore-scence-active quercetin (Qct) with casein (Cas) and bovine serum albumin (BSA) in phosphate buffer solution (PBS, pH=7.4) with or without coexisting carbon nanotubes (CNTs). Formulae for binding constant (K) and molar binding ratio (n) were established for methods 1 (fixing protein concentration, changing Qct concentration, and monitoring the fluorescence of protein) and 2 (fixing Qct concentration, changing protein concentration, and monitoring the fluorescence of Qct), to which values of K and n were calculated via nonlinear least-square fitting of the experimental data, and the “optical inner filtering induced fluorescence quenching” effect was thus quantitatively evaluated. The quenching effects of coexisting CNTs on the fluorescence of Qct, BSA, and Cas, as well as the effects of coexisting CNTs on Qct-BSA and Qct-Cas interactions, were examined. Synchronous fluorescence was also used to examine the effects of coexisting CNTs and Qct on the conformations of BSA and Cas, with relevant K and n values for tyrosine (Tyr) and tryptophan (Trp) residues estimated. It was concluded that the CNTs mainly interacted with the Trp residues locating near the protein surfaces, but small-sized Qct molecules could further interact with the Tyr residues locating inside the protein molecules.     

溴百里酚蓝与牛血清白蛋白的相互作用研究

在模拟动物体生理条件和不同温度下,用荧光光谱和紫外-可见吸收光谱法研究了溴百里酚蓝(BTB)与牛血清白蛋白(BSA)结合反应的光谱行为。用Stern-Volmer和Lineweaver-Burk方程分别处理试验数据,发现BSA与BTB发生反应生成了新的复合物,属于静态荧光猝灭。求出了反应时复合物的形成常数KLB(2.792×105L.mol-1)、热力学参数(ΔHθ=(20.24 kJ.mol-1,ΔSθ=37.22J.K-1,ΔGθ=(31.25kJ.mol-1)与结合位点数(1.1578)。根据F rster偶极-偶极非辐射能量转移理论计算出结合位置距离212位色氨酸残基2.60nm,证明二者主要靠静电作用力结合。同时用同步荧光光谱和三维荧光光谱法探讨了BTB对BSA构象的影响,表明BTB使色氨酸残基所处微环境的极性减弱、疏水作用增强,为阐明BTB的染色机理、毒理效应和生物学效应提供重要信息。     

重金属镉离子人工合成抗原的荧光特性分析

以1-(4-异硫氰苄基)乙烯基二胺-N,N,N′,N′-四乙酸为双功能螯合剂,偶联Cd2+与血清蛋白质分子, 合成了Cd2+人工抗原.利用荧光光谱分析了Cd2+人工合成抗原的荧光特性并得到合成反应的偶联比为11∶ 1～16∶ 1;通过荧光相图分析表明, Cd2+人工抗原合成过程中血清蛋白质的折叠状态符合"二态模型";偏振荧光光谱检测结果显示, Cd2+人工抗原合成中血清蛋白质色氨酸残基的微区构象发生一定变化,导致载体蛋白质构象改变.     

Interaction of Surface-active Fluorescence Probes with Bovine Serum Albumin

The binding between three surface-active substituted 3H-indole fluorescence probes and bovine serum albumin (BSA) in aqueous solution was studied using fluorescence quenching. The binding constants of 3H-indole molecules with BSA were obtained. According to the Foerster resonance energy transfer theory, the distances between 3H-indole molecules and tryptophan of BSA were calculated. The results show that the oligoethyloxyethylene chain of 3H-indole molecules is longer, the binding between them is stronger, the energy transfer efficiency is higher, and the distance between tryptophan and 3H-indole is nearer.     

三维同步偏振荧光光谱研究蛋白质溶液变性时氨基酸残基的发光行为

将偏振技术、同步技术与三维技术结合起来的三维同步偏振荧光光谱(TDSPS)能分辨蛋白质溶液中的色氨酸(Trp)和酪氨酸(Tyr)残基,具有同步光谱分辨率高、三维技术信息丰富的优点．本文用TDSPS表征牛血清白蛋白(BSA)、人血清白蛋白(HSA)受各种因素影响(酸效应、碱效应、盐效应、猝灭剂等)时Trp,Tyr残基荧光光谱的变化,用于区分HSA和BSA．     

A fluorescence spectroscopic study of a coagulating protein extracted from Moringa oleifera seeds

The fluorescence studies of coagulating protein extracted from Moringa oleifera seeds have been studied using steady-state intrinsic fluorescence. The fluorescence spectra are dominated by tryptophan emission and the emission peak maximum (lambda(max)=343+ or -2nm) indicated that the tryptophan residue is not located in the hydrophobic core of the protein. Changes in solution pH affected the protein conformation as indicated by changes in the tryptophan fluorescence above pH 9 whereas the ionic strength had minimal effect. The exposure and environments of the tryptophan residue were determined using collisional quenchers.     

桑色素与血清白蛋白相互作用热力学行为

利用荧光光谱、紫外-可见吸收光谱、圆二色谱、分子模拟等方法,在近似生理条件下,以牛血清白蛋白(BSA)为模式蛋白质,研究了桑色素(Morin)和血清白蛋白相互作用的热力学行为及其特征。荧光光谱结果表明:Morin能有效猝灭BSA的内源荧光,猝灭机制为静态猝灭;通过van’t Hoff方程,获取了BSA与Morin结合的热力学参数(?H?、?S?、?G?等),发现Morin与BSA两者之间的相互作用是一个吉布斯自由能降低的自发过程,且氢键和范德华力是二者结合的驱动力。通过分子模拟方法,发现Morin结合在BSA分子亚结构域IIIA的疏水腔内位点II,荧光共振能量转移结果表明Morin和与BSA的两个色氨酸残基的平均距离为3.09nm。圆二色谱结果表明Morin分子的结合会引起BSA分子α-螺旋含量降低。     

Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants

Bovine (BSA) and human (HSA) serum albumins are frequently used in biophysical and biochemical studies since they have a similar folding, a well known primary structure, and they have been associated with the binding of many different categories of small molecules. One important difference of BSA and HSA is the fact that bovine albumin has two tryptophan residues while human albumin has a unique tryptophan. In this work results are presented for the interaction of BSA and HSA with several ionic surfactants, namely, anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), as monitored by fluorescence spectroscopy of intrinsic tryptophans and circular dichroism spectroscopy. On the interaction of all three surfactants with BSA, at low concentrations, a quenching of fluorescence takes place and Stern-Volmer analysis allowed to estimate their 'effective' association constants to the protein: for SDS, CTAC and HPS at pH 7.0 these constants are, respectively, (1.4+/-0.1) x 10(5) M(-1), (8.9+/-0.1) x 10(3) M(-1) and (1.4+/-0.1) x 10(4) M(-1). A blue shift of maximum emission is observed from 345 to 330 nm upon surfactant binding. Analysis of fluorescence emission spectra allowed to separate three species in solution which were associated to native protein, a surfactant protein complex and partially denatured protein. The binding at low surfactant concentrations follows a Hill plot model displaying positive cooperativity and a number of surfactant binding sites very close to the number of cationic or anionic residues present in the protein. Circular dichroism data corroborated the partial loss of secondary structure upon surfactant addition showing the high stability of serum albumin. The interaction of the surfactants with HSA showed an enhancement of fluorescence at low concentrations, opposite to the effect on BSA, consistent with the existence of a unique buried tryptophan residue in this protein with considerable static quenching in the native state. The effects of surfactants at low concentrations were very similar to those of myristic acid suggesting a non specific binding through hydrophobic interaction modulated by eletrostatic interactions. The changes in the vicinity of the tryptophan residues are discussed based on the recently published crystallographic structure of HSA myristate complex (S. Curry et al., Nat. Struct. Biol. 5 (1998) 827).