铜(Ⅱ)离子与神经红蛋白的相互作用

利用紫外可见吸收光谱、荧光光谱、同步荧光光谱及圆二色(CD)光谱研究了铜髤离子与神经红蛋白(NGB)的相互作用。结果表明,Cu2+离子使NGB在280nm处的紫外吸收增强,说明Cu2+与NGB发生了相互作用;Cu2+使NGB内源性荧光发生猝灭,其猝灭机制为静态猝灭;同步荧光光谱表明,Cu2+使色氨酸微环境的疏水性有所降低,Cu2+对NGB的作用位点更接近于色氨酸;CD光谱显示Cu2+没有引起NGB二级结构明显的变化。     

Multi-spectroscopic Investigations of Aspirin and Colchicine Interactions with Human Hemoglobin: Binary and Ternary Systems

The interactions of colchicine (COL) and aspirin (ASA) with human hemoglobin (HB) was studied by fluorescence, UV/vis absorption, resonance light scattering, synchronous fluorescence and circular dichroism (CD) spectroscopic techniques under physiological conditions. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were determined by the fluorescence quenching technique at different temperatures. The results proved that the mechanism of fluorescence quenching of HB by COL and ASA is due to formation of HB–drug complexes in the binary and ternary systems. The distance between the acceptor drugs and HB was estimated by Förster’s equation on the basis of fluorescence energy transfer. In addition, according to the synchronous fluorescence spectra of HB, the results showed that the fluorescence quenching of HB originated solely from the tryptophan residues and indicated a conformational change for HB caused by addition of the drugs. Far-UV CD spectra of HB were recorded before and after the addition of ASA and COL both as binary and ternary systems. An increase in intensity of the positive CD peak of HB was observed in the presence of these drugs. The results were interpreted as excited state interactions between the aromatic residues of the HB binding sites and the drugs bound to them.     

Studies on the interaction of salvianolic acid B with human hemoglobin by multi-spectroscopic techniques

The interaction between salvianolic acid B (Sal B) and human hemoglobin (HHb) under physiological conditions was investigated by UV-vis absorption, fluorescence, synchronous fluorescence and circular dichroism spectroscopic techniques. The experimental results indicate that the quenching mechanism of fluorescence of HHb by Sal B is a static quenching procedure, the binding reaction is spontaneous, and the hydrophobic interactions play a major role in binding of Sal B to HHb. Based on F?rster's theory of non-radiative energy transfer, the binding distance between Sal B and the inner tryptophan residues of HHb was determined to be 2.64 nm. The synchronous fluorescence experiment revealed that Sal B can not lead to the microenvironmental changes around the Tyr and Trp residues of HHb, and the binding site of Sal B on HHb is located at α(1)β(2) interface of HHb. Furthermore, the CD spectroscopy indicated the secondary structure of HHb is not changed in the presence of Sal B.     

Study on the interaction of puerarin with lysozyme by spectroscopic methods

The interaction between lysozyme (LYSO) and puerarin has been studied at three temperatures (294, 302 and 310K) through/using fluorescence spectroscopy and circular dichroism (CD). The LYSO fluorescence was quenched by the binding of puerarin to LYSO. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. According to the van't Hoff equation, the standard enthalpy change (DeltaH degrees ) and standard entropy change (DeltaS degrees ) for the reaction were calculated to be 17.47kJ/mol and 163.5J/molK. It indicated that the hydrophobic interactions play a main role in the binding of puerarin to LYSO. In addition, the distance between puerarin (acceptor) and tryptophan residues of LYSO (donor) was estimated to be 1.47nm on the basis of fluorescence energy transfer. The changes of LYSO secondary structure in the presence of puerarin were observed from CD spectroscopy.     

CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α-, β-, and γ-, was studied by using fluorescence and circular dichroism techniques. α-Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α-crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H₂O₂-mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α-crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.     

人纤维蛋白溶酶原中色氨酸残基的化学修饰

以Ｎ－溴代琥珀酰亚胺为修饰剂，对人纤维蛋白溶酶原（ＨＰｇ）中色氨酸（Ｔｒｐ）残基的分布及其与酶活力的关系进行了研究，发现每个ＨＰｇ分子有１９个Ｔｒｐ残基，５个位于分子表面：有２个是快反应残基，其中１个是活性必需的氨基酸，酶被修饰后其荧光光谱及圆二色谱发生了变化。     

氯硝柳胺及其衍生物与钥孔戚血蓝蛋白的相互作用

以氯硝柳胺为原料合成了聚乙二醇200基氯硝柳胺. 采用荧光光谱、同步荧光光谱、紫外吸收光谱和圆二色谱研究了氯硝柳胺及其衍生物与钥孔戚血蓝蛋白(KLH)的相互作用. 结果表明, 两种药物分子对KLH的荧光猝灭机制属于静态猝灭; 由Lineweaver-Burk方程计算出不同温度下结合常数K, 但是聚乙二醇200基氯硝柳胺与KLH的作用相对较弱; 由Van′t Hoff方程计算出ΔH和ΔS平均值, 结合力主要为静电作用力; 热力学函数计算结果表明, 氯硝柳胺及其衍生物与KLH的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; 根据Förster非辐射能量转移机制求得给体与受体间的结合距离r均小于7 nm; 同步荧光光谱表明, 氯硝柳胺及其衍生物能够被血蓝蛋白存储和转运, 但结合时对蛋白构象有一定影响; 圆二色谱测得加入两种药物后, KLH的α-螺旋含量均降低, 二级结构发生改变. 通过比较氯硝柳胺及其衍生物与KLH的相互作用, 初步探讨了分子结构与其结合能力之间的联系.     

光谱法研究Cu2+与肌红蛋白的相互作用

用紫外吸收光谱、荧光光谱、同步荧光光谱及圆二色(CD)谱研究了Cu2+与肌红蛋白(Mb)的相互作用. 结果发现, Cu2+使Mb的紫外吸收增强, 峰位蓝移, 说明Cu2+与Mb发生了较强的相互作用; Mb的特征荧光峰猝灭, 且随着温度升高猝灭常数Ksv降低, 表明Cu2+对Mb的荧光猝灭机制属于静态猝灭; 计算了不同温度下的结合常数和结合位点数; 由van′t Hoff方程计算出ΔH和ΔS分别为-11.60 kJ/mol和33.77 J·(mol·K)-1, 得出二者之间的作用力主要为静电力; 并依据Förster非辐射能量转移理论确定了给体-受体间的结合距离r=2.56 nm. 同步荧光光谱表明, Cu2+对Mb的构象产生影响, 使色氨酸残基的疏水性下降. CD光谱测得加入Cu2+后, 二级结构发生改变, 使α-螺旋含量降低.     

Use of spectroscopic, zeta potential and molecular dynamic techniques to study the interaction between human holo-transferrin and two antagonist drugs: comparison of binary and ternary systems

For the first time, the binding of ropinirole hydrochloride (ROP) and aspirin (ASA) to human holo-transferrin (hTf) has been investigated by spectroscopic approaches (fluorescence quenching, synchronous fluorescence, time-resolved fluorescence, three-dimensional fluorescence, UV-vis absorption, circular dichroism, resonance light scattering), as well as zeta potential and molecular modeling techniques, under simulated physiological conditions. Fluorescence analysis was used to estimate the effect of the ROP and ASA drugs on the fluorescence of hTf as well as to define the binding and quenching properties of binary and ternary complexes. The synchronized fluorescence and three-dimensional fluorescence spectra demonstrated some micro-environmental and conformational changes around the Trp and Tyr residues with a faint red shift. Thermodynamic analysis displayed the van der Waals forces and hydrogen bonds interactions are the major acting forces in stabilizing the complexes. Steady-state and time-resolved fluorescence data revealed that the fluorescence quenching of complexes are static mechanism. The effect of the drugs aggregating on the hTf resulted in an enhancement of the resonance light scattering (RLS) intensity. The average binding distance between were computed according to the forster non-radiation energy transfer theory. The circular dichroism (CD) spectral examinations indicated that the binding of the drugs induced a conformational change of hTf. Measurements of the zeta potential indicated that the combination of electrostatic and hydrophobic interactions between ROP, ASA and hTf formed micelle-like clusters. The molecular modeling confirmed the experimental results. This study is expected to provide important insight into the interaction of hTf with ROP and ASA to use in various toxicological and therapeutic processes.     

大黄酸铜(Ⅱ)配合物与血清白蛋白的相互作用

采用荧光光谱法和紫外光谱法研究了大黄酸铜配合物与牛血清白蛋白之间的相互作用.大黄酸铜配合物能显著猝灭牛血清白蛋白的内源荧光并以静态猝灭为主;计算了298 K和309 K温度下结合常数、结合位点,根据热力学参数判断大黄酸铜配合物与牛血清白蛋白之间具有较强的疏水作用力;依据F?rster的偶极-偶极非辐射能量转移理论,计算出大黄酸铜在蛋白质中结合位置与色氨酸残基间的距离为3.21 nm, 表明大黄酸铜的部分片段能够插入蛋白质分子内部;用同步荧光光谱和圆二色光谱技术探讨了大黄酸铜对牛血清白蛋白构象的影响.     

Spectroscopic studies on the interaction between human hemoglobin and CdS quantum dots

The interaction between human adult hemoglobin (Hb) and bare CdS quantum dots (QDs) was investigated by fluorescence, synchronous fluorescence, circular dichroism (CD), and Raman spectroscopic techniques under physiological pH 7.43. The intrinsic fluorescence of Hb is statically quenched by CdS QDs. The quenching obeys the Stern-Volmer equation, with an order of magnitude of binding constant (K) of 10(7). The electrostatic adsorption of Hb on the cationic CdS QDs surface is energetically favorable (DeltaS(0)=70.22 Jmol(-1)K(-1), DeltaH(0)=-23.11 kJmol(-1)). The red shift of synchronous fluorescence spectra revealed that the microenvironments of tryptophan and tyrosine residues at the alpha(1)beta(2) interface of Hb are disturbed by CdS QDs, which are induced from hydrophobic cavities to a more exposed or hydrophilic surrounding. The secondary structure of the adsorbed Hb has a loose or extended conformation for which the content of alpha-helix has decreased from 72.5 to 60.8%. Moreover, Raman spectra results indicated that the sulfur atoms of the cysteine residues form direct chemical bonds on the surface of the CdS QDs. The binding does not significantly affect the spin state of the heme iron, and deoxidation is not expected to take place on the coated oxyhemoglobin. The change of orientation of heme vinyl groups was also detected.     

羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响

在模拟生理条件下, 利用荧光光谱、 圆二色光谱和紫外-可见吸收光谱探究了羟丙基-β-环糊精(HPCD)对1-羟基芘(1-OHPyr)与牛血清白蛋白(BSA)相互作用的影响及其相关机制. 结果表明, 291 K下HPCD可使1-OHPyr与BSA的结合常数降低为1.45×10⁵ L/mol; 导致1-OHPyr-BSA体系中BSA的α-螺旋含量恢复, 色氨酸(TRP)残基周围微环境的极性变化减弱; 致使1-OHPyr及BSA的荧光寿命均延长, 且二者的结合距离增大. 初步研究结果表明, HPCD与1-OHPyr的包络作用是HPCD影响BSA与1-OHPyr结合的主要原因.     

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).     

果菠萝蛋白酶在有机溶剂微扰时的分子折叠与活力变化的研究

本文用荧光、紫外差示及CD光谱研究果菠萝蛋白酶经甲醇、乙醇、乙二醇微扰后的构象与活力变化情况.酶的荧光强度随有机溶剂浓度增大而增强,表明Tyr、Trp微环境发生明显变化。232nm和285nm处出现紫外差吸收正峰。前峰与酶分于折叠的变化有关,而后峰与Tyr、Trp微环境的变化相关.甲醇、乙醇微扰后,天然酶的208nm和225nmCD双负峰逐渐加强,而乙二醇微扰后,225nm负峰加强。208nm负峰减弱并红移直至完全消失,说明酶分子完全伸展.     

Binding analysis of farrerol to lysozyme by spectroscopic methods

Binding of farrerol to lysozyme (LYSO) was investigated at 302, 313 and 318 K at pH 7.4 using spectrophotometric techniques such as fluorescence emission, circular dichroism (CD) and UV absorption. The data obtained from fluorescence quenching experiments showed that farrerol was bound to LYSO and the affinity was enhanced by the addition of farrerol. When the concentration ratio of farrerol to LYSO was higher than 5.4, both the binding constant and the binding stoichiometry went up. Based on the thermodynamic parameters evaluated from the van't Hoff equation, the enthalpy change (deltaH degrees ) and entropy change (deltaS degrees ) were derived to be negative values. They indicated that both van der Waals forces and hydrogen bonds are the major interactions between farrerol and LYSO. A value of 2.67 nm for the average distance r between farrerol (acceptor) and tryptophan residues (Trp) of LYSO (donor) was derived from the fluorescence resonance energy transfer. Besides, the change in the conformation of LYSO was observed, being caused by the interaction with farrerol.     

羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响

在模拟生理条件下,利用荧光光谱、圆二色光谱和紫外-可见吸收光谱探究了羟丙基-β-环糊精(HPCD)对1-羟基芘(1-OHPyr)与牛血清白蛋白(BSA)相互作用的影响及其相关机制.结果表明,291 K下HPCD可使1-OHPyr与BSA的结合常数降低为1.45×10~5L/mol;导致1-OHPyr-BSA体系中BSA的α-螺旋含量恢复,色氨酸(TRP)残基周围微环境的极性变化减弱;致使1-OHPyr及BSA的荧光寿命均延长,且二者的结合距离增大.初步研究结果表明,HPCD与1-OHPyr的包络作用是HPCD影响BSA与1-OHPyr结合的主要原因.     

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。     

Studies on the interaction of cinnamic acid with bovine serum albumin

The interaction between cinnamic acid and bovine serum albumin (BSA) have been studied at three temperatures, 296, 303 and 310 K. Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of cinnamic acid in aqueous solution at pH 7.40. The fluorescence quenching constant K(q), K(sv) and the binding constant K were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of cinnamic acid. The thermodynamic parameters, the enthalpy (DeltaH) and the entropy change (DeltaS) were estimated to be -16.457 kJ mol(-1) and 38.028 J mol(-1) K(-1) according to the van't Hoff equation. The displacement experiment shows that cinnamic acid can bind to the subdomain IIA (corresponding to Sudlow's drug binding site I). The distance between the tryptophan residues in BSA and cinnamic acid bound to site I was estimated to be 1.63 nm using F?ster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of common ions indicates that common ions have effect on drug-BSA system.     

Hemin和CopC相互作用的光谱和分子模拟研究

本文通过光谱技术以及分子模拟手段研究了CopC与hemin的相互作用。紫外可见光谱表明hemin中Fe³⁺可能与CopC中的组氨酸(His1或His91)形成配位。此外,荧光光谱表明CopC与hemin的主要弱作用力为疏水作用力。圆二色谱、化学变性、荧光寿命以及同步荧光实验表明hemin与CopC的配位使得CopC的稳定性降低,并且色氨酸周围环境的疏水性减弱。紫外可见及荧光光谱表明,两者结合常数在10⁶左右,结合比为1,hemin与CopC中唯一的色氨酸(Trp83)的距离为1.06 nm。最后通过分子对接模拟了CopC与hemin的相互作用,结果表明hemin中Fe³⁺可能与CopC中的组氨酸(His1)形成与卟啉环平面垂直的轴向配位,并且与Trp83距离为0.96 nm,与实验结果一致。     

牛血红蛋白与银纳米粒子相互作用的光谱研究

运用紫外-可见吸收光谱(UV-Vis)、荧光光谱、同步荧光光谱、圆二色光谱(CD)及傅立叶变换红外光谱(FTIR)等手段, 研究牛血红蛋白(Bovine Hemoglobin, 简称BHb)与银纳米粒子的相互作用. 结果表明, BHb能吸附在银纳米粒子的表面, 使其415 nm处的特征等离子体共振吸收峰强度下降, 峰位红移. 随银纳米粒子的浓度增大, BHb分子中Soret带的吸收持续降低, 说明银纳米粒子可能使部分血红素辅基从它们的键腔中脱离出来. Stern-Volmer方程分析表明, 银纳米粒子静态猝灭BHb的内源荧光. 由UV-Vis和荧光光谱的变化, 计算BHb与银纳米粒子的结合常数, 其数量级达到10⁹～10¹⁰. 同步荧光光谱的蓝移说明, 银纳米粒子扰动BHb分子内部的酪氨酸、色氨酸残基所处的微环境, 使之包埋于疏水腔中. 拟合计算远紫外CD数据发现, 银纳米粒子诱导BHb产生轻微的二级结构改变, α-螺旋含量降低. FTIR光谱结果提示, BHb中半胱氨酸残基的硫、羧基氧、酰胺及氨基酸残基中的氮原子与银纳米粒子可能有表面键合作用.