秋水仙碱与人血清白蛋白相互作用的谱学研究

采用紫外、荧光和圆二色光谱研究了秋水仙碱与人血清白蛋白之间的相互作用.结果发现秋水仙碱使人血清白蛋白的紫外吸收增强,特征荧光峰猝灭,并且随温度升高猝灭常数Ksv降低.求算了不同温度下秋水仙碱与人血清白蛋白相互作用的平衡常数与结合位点数.根据Van't Hoff方程计算出△H=-11.66kJ·mol-1,△S=51.507 J·(mol·K)-1,得出二者之间的作用力主要是静电作用力.圆二色光谱测得加入秋水仙碱后人血清白蛋白的α-螺旋降低,二级结构改变,表明秋水仙碱对人血清白蛋白的荧光猝灭机制属于形成配合物所引起的静态猝灭.     

芦丁与血清白蛋白结合作用的热力学研究(I)

在生理pH值条件下,研究了芦丁与牛血清白蛋白和人血清白蛋白之间的结合作用。通过荧光法确定了芦丁与血清白蛋白的荧光猝灭机制,根据热力学方程讨论了两者间的主要作用力类型。芦丁对血清白蛋白的荧光猝灭机制为静态猝灭。确定了不同温度下该结合反应的结合常数和结合位点数,并根据热力学方程求得了结合反应的热力学参数。两者结合的主要作用力类型是氢键和Vander Waals力。芦丁在体内能够被血清白蛋白存储和转运,且结合时对蛋白质构象无影响。     

荧光猝灭法研究刚果红与人血清白蛋白的相互作用

在0.050mol.L-1的Tris-HCl缓冲介质中（内含0.10mol.L-1NaCl）,用荧光光谱法在模拟生理条件下,研究了刚果红与人血清白蛋白的相互作用。在不同温度和不同pH值下,刚果红对人血清白蛋白的荧光猝灭作用为静态猝灭机制。根据荧光猝灭双倒数曲线和位点结合模型计算出的刚果红与人血清白蛋白之间的结合常数相当,且结合常数与结合位点数都随温度升高而减小,在pH7.4时最大。根据热力学方法讨论了两者间主要的作用力类型,由重叠积分面积,得出两者的结合距离。由此可见,刚果红与人血清白蛋白之间有很强的结合作用。     

光谱法研究盐酸非那吡啶与牛血清白蛋白的结合作用

运用光谱学方法研究了在生理pH值条件下盐酸非那吡啶(PHE)与牛血清白蛋白之间的结合作用.通过荧光光谱和紫外吸收光谱确定了盐酸非那吡啶对牛血清白蛋白的荧光猝灭机理.依据Scatchard方程测定了不同温度下该结合反应的结合常数和结合位点数.根据热力学方程讨论了两者间的主要作用力类型.结合同步荧光光谱分析了盐酸非那吡啶对牛血清白蛋白构象的影响.盐酸非那吡啶对牛血清白蛋白的荧光猝灭机制主要为静态猝灭和非辐射能量转移.在15,25,37℃时盐酸非那吡啶与牛血清白蛋白的结合常数Kb分别为2.47×107,9.15×106,4.36×106 L·mol-1,它们之间平均结合位点数n为1.结合反应的热力学参数为△H=-71.2 kJ·mol-1,△S=124.8 J·mol-1·K-1.热力学函数计算结果表明,该作用过程是一个熵增加,Gibbs自由能降低的自发分子间作用过程.依据F(o)rster能量转移理论确定PHE与BSA间的结合距离为1.61 nm.两者结合的主要作用力类型是静电作用力.盐酸非那吡啶在体内能够被血清蛋白存储和转运,但结合时对蛋白构象有一定影响.     

荧光法研究左氧氟沙星与人血清白蛋白作用机理及含量测定

利用荧光及紫外光谱法研究了水溶液体系中左氧氟沙星(LVFX)与人血清白蛋白(HSA)的相互作用机制.结果表明左氧氟沙星对人血清白蛋白的荧光有较强的猝灭作用,其猝灭类型主要为静态猝灭.在不同温度下求得了左氧氟沙星与人血清白蛋白的结合常数k.发现随反应温度上升k值下降.由热力学参数.确定了左氧氟沙星与人血清白蛋白的结合作用主要为色散力.用同步荧光技术考察了左氧氟沙星对人血清白蛋白构象的影响.又根据Foerster理论,测得了左氧氟沙星与入血清白蛋白结合的能量转移效率.相互结合距离.进一步证明了该反应是单一静态猝灭过程.阐述了其猝灭机理是通过能量转移产生的.研究发现人血清白蛋白荧光猝灭程度的对数与左氧氟沙星含量有良好的线性关系,25 C时线性范围为:2.O×10-6-2.4×10-5mol·L-1,检出限为1.2×10-7mol·L-1.用于片剂中左氧氟沙星含量的测定结果满意,相对标准偏差为:1.5%-2.1%,回收率为:94.8%-97.7%.     

4-硫胸苷的合成及其与人血清白蛋白的相互作用

基于含硫核苷类化合物的光敏性和核苷类化合物对肿瘤细胞有特殊的亲和力以及核苷类化合物的抗肿瘤活性,设计合成了4-硫胸苷,并通过NMR、UV、MS对其结构进行了表征。利用荧光光谱考察了4-硫胸苷与人血清白蛋白(HSA)的相互作用。实验结果表明:4-硫胸苷对人血清白蛋白(HSA)体系的猝灭是由于他们相互作用形成复合物而导致的静态猝灭。通过计算得出其结合常数分别为3.46×104,3.91×104,4.31×104L·mol-1,并测定了不同温度下的热力学参数。结果表明,4-硫胸苷与人血清白蛋白之间的主要作用力类型为疏水作用力。另外,通过圆二色光谱考察了4-硫胸苷对人血清白蛋白二级结构的影响,结果表明,4-硫胸苷对人血清白蛋白的二级结构基本没有影响。     

2,4-二氯苯酚与人血清白蛋白的相互作用研究

通过2,4-二氯苯酚与人血清白蛋白结合反应的光谱学特征,探讨2,4-二氯苯酚与人血清白蛋白的结合反应机制;应用荧光光谱法和吸收光谱法,根据荧光猝灭数据,由Stern-Volmer方程和采用lg[(F0-F)/F]对lg[Q]作图,进行一元线性回归处理求出2,4-DCP与HSA的猝灭常数(Kq)、结合常数(Ksv)和结合位点数(n)及反应热力学参数;荧光静态猝灭常数Kq=3.713×1013L·mol-1·s-1,结合常数为2.743×106L/mol、结合位点数为n=1;2,4-二氯苯酚对人血清白蛋白内源荧光的猝灭机制,主要是通过疏水作用力,形成了无荧光特征的复合物所引起的静态荧光猝灭。     

荧光光谱法研究2-硝基苯胺与牛血清白蛋白的相互作用

采用荧光光谱法研究了不同酸度下,2-硝基苯胺与牛血清白蛋白(BSA)间的相互作用。实验结果表明,2-硝基苯胺对BSA的猝灭机制属于静态猝灭过程。经研究得到了不同酸度和不同温度下2-硝基苯胺与BSA反应的结合常数、结合位点数。根据热力学常数确定了二者间的作用力类型为氢键和疏水作用力。同步荧光结果表明,2-二硝基苯胺的存在改变了牛血清白蛋白的分子构象。     

白藜芦醇与人血清白蛋白相互作用的光谱研究

在不同温度下,用荧光猝灭光谱、同步荧光光谱和紫外一可见吸收光谱,研究了白藜芦醇与人血清白蛋白(HSA)相互作用的光谱学行为.根据不同温度下白藜芦醇对HSA的荧光猝灭作用,利用Stern-Volmer方程处理实验数据,结果表明白藜芦醇与}tSA的结合常数KA为2.39×105(25℃),1.25×105(35℃)和1.10×10 5(45℃).根据Forster非辐射能量转移理论,求出了白藜芦醇与HSA之间的结合距离为3.02 nm(25℃),3.46nm(35℃)和3.79 nm(45℃).实验表明静态猝灭和非辐射能量转移是导致白藜芦醇对HSA荧光猝灭的两大原因,通过计算热力学参数,可知该药物与人血清白蛋白的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的主要作用力类型为疏水作用力.并采用同步荧光光谱探讨了白藜芦醇对HSA构象的影响.     

山姜素与人血清白蛋白相互作用的荧光光谱法研究

利用荧光光谱法和紫外-可见光谱法研究了山姜素与人血清白蛋白(HSA)之间的相互作用。证实了山姜素对HSA的荧光猝灭为动态猝灭过程,并测定了不同温度下的猝灭常数;根据Frster非辐射能量转移理论,计算出山姜素在蛋白质中的结合位置与色氨酸残基间的距离为4.05nm;由求得的热力学参数,推断了山姜素与HSA之间主要靠疏水作用力结合;用三维荧光光谱及同步荧光光谱技术探讨了山姜素对HSA构象的影响。     

Transport of a Cancer Chemopreventive Polyphenol,Resveratrol: Interaction with Serum Albumin and Hemoglobin

Resveratrol is a natural phytoalexin with pharmacologic effects on several human diseases: carcinogenesis, coronary heart disease and neurodegenerative disease. Due to its poor water solubility, resveratrol must be bound to proteins to keep it at a high concentration in serum. In our work, the bindings of resveratrol to plasma proteins, human serum albumin (HSA) and hemoglobin (Hb), have been investigated systematically by fluorescence quenching technique, synchronous fluorescence, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. The fluorescence data show that the binding of resveratrol to HSA or Hb is a static quenching procedure and each protein has only one binding site for the drug. The binding constant of resveratrol to HSA is larger than that of resveratrol to Hb at corresponding temperature, which indicates that the affinity of HSA toward the drug is higher than that of Hb. The CD spectroscopy indicates that the secondary structures of the proteins are changed in the presence of resveratrol with the reduction of α-helices, which decreased about 18.75% for HSA and 9.43% for Hb at the drug to proteins molar ratio of 2. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction plays a major role in the binding of resveratrol to HSA, and hydrogen bonding is the mainly binding force in the binding of resveratrol to Hb. The study of molecular modeling shows that resveratrol is located in the hydrophobic cavity between subdomain IB and IIA of HSA (the entrance of site I), or located in the central cavity of Hb (partial to the subunit A).     

Study on the interaction between tabersonine and human serum albumin by optical spectroscopy and molecular modeling methods

The mechanism of interaction between tabersonine (TAB) and human serum albumin (HSA) was investigated by the methods of fluorescence spectroscopy, UV–vis absorption spectroscopy and molecular modeling under simulative physiological conditions. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that TAB has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding site number n and apparent binding constant K_a, corresponding thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were calculated. The distance r between donor (human serum albumin) and acceptor (tabersonine) was obtained according to the Förster theory of non-radiation energy transfer. The effect of common ions on binding constant was also investigated. The synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of HSA molecules with addition of TAB. Furthermore, the study of molecular modeling indicated that TAB could bind to the site I of HSA and hydrophobic interaction was the major acting force, which was in agreement with the binding mode study.     

Complexes between C.I. Acid Orange 6 and human serum albumin, a multi-spectroscopic approach to investigate the binding behavior

The interaction mechanism of Acid Orange 6 (AO6) with human serum albumin (HSA) was investigated firstly by using fluorescence quenching technique, UV absorbance, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence spectroscopy in combination with molecular modeling method under simulative physiological conditions. Fluorescence data indicated that there is a single class of binding sites between AO6 and HSA, and the alterations of HSA secondary structure in the presence of AO6 was confirmed by synchronous fluorescence, UV, CD, FT-IR and three-dimensional fluorescence spectra. The efficiency of fluorescence resonance energy transfer provided the binding distance (r) of 2.83 nm for AO6-HSA system. Furthermore, the thermodynamic parameters enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were calculated to be −5.77 kJ mol⁻¹ and 109.42 J mol⁻¹ K⁻¹, respectively, according to Van't Hoff equation, these data suggested that both hydrophobic forces and hydrogen bonding play a major role in the binding of AO6 to HSA, which agrees well with the results of molecular modeling study. Experimental results showed that the interaction between AO6 and HSA induced a conformational change of HSA, which was proved by the qualitative and quantitative analysis data of different spectroscopic techniques under simulative physiological conditions.     

Characterization of Interaction Between Bergenin and Human Serum Albumin in Membrane Mimetic Environments

The interaction between bergenin and human serum albumin (HSA) in AOT/isooctane/water microemulsions was studied by fluorescence quenching technique in combination with UV absorption spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique. Fluorescence data in omega (o) 20 microemulsions revealed the presence of a binding site of bergenin on HSA and its binding constants (K) were 1.64 x 10(4), 1.44 x 10(4), 1.26 x 10(4) and 1.09 x 10(4) M(-1) at 289, 296, 303, and 310 K, respectively. The binding of bergenin with HSA in microemulsions was stronger than that in buffer solution. The alterations of protein secondary structure in the microemulsions in the absence and presence of bergenin compared with the free form of HSA in buffer were qualitatively and quantitatively analyzed by the evidence from CD spectra. Enthalpy and entropy changes for the reaction were calculated to be -14.45 kJ mol(-1) and 30.76 J mol(-1) K(-1). These results indicated that bergenin bound to HSA mainly by a hydrophobic interaction in microemulsions which was in agreement with the result of the molecular modeling study. The DLS data suggested that HSA may locate at the interface of the microemulsion and bergenin could interact with them.     

多光谱和分子模拟技术研究全氟十二酸与人血清白蛋白的相互作用

全氟十二酸（PFDoA）是8～12个碳链的全氟烷酸（PFAAs）中毒性最强的新型环境污染物。已有大量研究表明PFAAs在环境中广泛积累,但对PFDoA与HSA的相互作用还处于起步阶段。本研究力争在模拟生理条件下,采用荧光猝灭法、分子模拟技术和圆二色谱确定 HSA与 PFDoA的相互作用机理。研究结果表明,PFDoA对HSA的猝灭是动态猝灭与形成 PFDoA‐HSA基态复合物引起的猝灭共同作用的结果。计算得到的结合距离（r＝3.65 nm）表明,PFDoA（受体）与HSA（供体）之间的相互作用发生了非辐射能量转移。取代反应结果表明,PFDoA键合在HSA的site Ⅰ位点上。分子对接进一步研究了PFDoA与HSA作用的详细结合情况,表明PFDoA通过多种作用力结合在 HSA的亚域IIA内,例如,PFDoA上的O 1原子主要通过极性键与HSA上的Arg 257和Ser 287残基结合。计算得到的最优对接能量为－25.87 kJ · mol－1,表明PFDoA对 HSA有较大的结合亲和力。同步荧光光谱和三维荧光光谱研究了PFDoA对 HSA构象的影响,结果显示,与PFDoA结合后,色氨酸的微环境疏水性增加,HSA的构象也发生改变。PFDoA与 HSA作用前后圆二色谱二级结构的定量分析结果表明,PFDoA‐HSA复合物的形成使螺旋稳定性降低。该研究结果为全氟烷酸与HSA的动力学研究提供了理论依据和可靠数据,并揭示了生物大分子与配体相互作用的化学本质。     

The binding of 3-(p-bromophenyl)-5-methyl-thiohydantoin with human serum albumin: Investigation by fluorescence spectroscopy and molecular model

The binding of 3-(p-bromophenyl)-5-methyl-thiohydantoin (BPMT) with human serum albumin (HSA) was investigated by fluorescence spectroscopy in combination with UV absorption spectrum under physiological conditions. The intrinsic fluorescence of HSA was quenched by BPMT through static quenching mechanism and the fluorescence emission spectrum of HSA exhibited appreciable hypsochromic shift with increasing concentration of BPMT. The binding constants (K) of HSA with BPMT and the number binding sites (n) at different temperatures, thermodynamic parameter enthalpy changes (ΔH) and entropy changes (ΔS) of HSA-BPMT have been calculated according to the relevant fluorescence data, indicating that the hydrophobic interaction played a major role, which was consistent with the result of molecular modeling study.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

Combined molecular docking and multi-spectroscopic investigation on the interaction between Eosin B and human serum albumin

The binding of Eosin B to human serum albumin (HSA) was studied using molecular docking, fluorescence, UV–vis, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The mechanism of interaction between Eosin B and HSA in terms of the binding parameters, the thermodynamic functions and the effect of Eosin B on the conformation of HSA were investigated. Protein-ligand docking study indicated that Eosin B bound to residues located in the subdomain IIA of HSA and Eosin B–HSA complex was stabilized by hydrophobic force and hydrogen bonding. In addition, fluorescence data revealed that Eosin B strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure. Furthermore, alteration of the secondary structure of HSA in the presence of the dye was conformed by UV–vis, FT-IR and CD spectroscopy.     

光谱法和分子对接方法研究罗利环素与人血清白蛋白的相互作用

在模拟人体生理条件下，应用多光谱法和分子对接技术对罗利环素(RTC)与人血清白蛋白(HSA)的相互作用进行了研究。实验通过消除内滤光来提高荧光数据的准确性。此外，荧光光谱和紫外光谱的结果表明RTC与HSA的猝灭方式是静态猝灭，且在298和310 K温度下的结合位点数分别为1.09和0.95。在两个不同温度下的结合常数分别为K_{298 K}=3.13×105 L·mol-1和K_{310 K}=0.70×105 L·mol-1。根据热力学参数的计算结果可知，RTC与HSA的结合作用力主要是氢键和范德华力。取代实验表明，RTC在HSA的Site Ⅰ，ⅡA子域上有一个结合位点。根据非辐射能量转移理论得到的RTC和HSA氨基酸残基间的结合距离为2.59 nm。三维荧光光谱表明RTC和HSA的相互作用改变了蛋白质的构象。此外，采用傅里叶变换红外光谱法对RTC与HSA作用前后HSA二级结构的变化进行了定量分析，结果表明，α-螺旋结构含量降低了8.4%，β-折叠含量从30.3%增加到31.4%，β-转角也从15.6%增加到了16.1%。分子对接进一步显示RTC通过氢键、疏水作用力、极性键等多种作用力与HSA的ⅡA子域上氨基酸残基相互作用。实验结果有助于从分子水平研究RTC和HSA的相互作用。     

Interaction of 1-cyanoethyl-5-chlorouracil with human and bovine serum albumins

The interactions of human (HSA) and bovine (BSA) serum albumins with 1-cyanoethyl-5-chlorouracil (CECU) were investigated by fluorescence spectroscopy, UV absorption spectroscopy, and molecular modeling methods under the simulated physiological conditions. The results of fluorescence measurements indicate that CECU has a strong ability to quench the intrinsic fluorescence of both HSA and BSA through a static quenching procedure. The binding constants (K) at different temperatures and thermodynamic parameters, enthalpy change (ΔH), and entropy change (ΔS) were calculated according to fluorescence data. The results show that hydrophobic interaction is a predominant intermolecular force for stabilizing the complex, which is in agreement with the results of molecular modeling study. The effect of some normal ions on the binding constants is also discussed. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 737–745, September–October, 2008.