光谱法和分子对接模拟技术研究托拉塞米与胃蛋白酶和胰蛋白酶的相互作用

托拉塞米(TOR)属于吡啶磺酰脲类袢利尿剂,被广泛有效地用于高血压,心脏衰竭,慢性肾功能衰竭和肝脏疾病的治疗。TOR在治疗过程中易引起的不良反应之一为轻微肠胃不适。然而,TOR与消化蛋白酶(胰蛋白酶和胃蛋白酶)分子间的相互作用鲜有报道。在模拟生理条件下,采用荧光光谱、紫外-可见吸收光谱、圆二色谱和分子对接技术研究了不同温度下托拉塞米(Torasemide, TOR)与胃蛋白酶(Pepsin)和胰蛋白酶(Trypsin)间的相互作用。所有荧光数据均进行了内滤光校正以获得更准确的结合参数。结果表明,TOR-Pepsin和TOR-Trypsin体系的猝灭常数(K_SV)均与温度呈负相关,说明TOR与Pepsin及Trypsin之间的作用机制均为静态荧光猝灭。利用紫外-可见吸收光谱、同步荧光光谱、3D荧光光谱和圆二色光谱法考查了TOR对Trypsin和Pepsin构象的影响。结果发现胃蛋白酶或胰蛋白酶中酪氨酸残基的极性改变较色氨基更明显,TOR可改变色氨酸残基的微环境并降低Trypsin和Pepsin中β-折叠结构,进而可能影响其生理功能。分子对接结果表明,TOR与Pepsin的结合位点位于由Asp-32和Asp-215组成的活性中心周围,从而抑制Pepsin活性。而TOR通过疏水作用力结合在Trypsin的口袋型底物结合位点(S1口袋),促进底物进入酶活性中心,最终表现为Trypsin活性升高。该研究探讨了TOR与胃蛋白酶和胰蛋白酶的结合作用和毒性机制,为TOR的安全使用提供重要依据。     

荧光光谱法和分子对接模拟技术研究白藜芦醇与胃蛋白酶的相互作用

白藜芦醇（Resveratrol, RES）属于非黄酮类多酚化合物,存在于葡萄科、百合科等多种植物体内,是一种具有多种生物活性和药理作用的天然活性物质,被广泛应用于食品和药品领域。研究表明多酚在生物体消化吸收过程中,会与消化酶（如胃蛋白酶、胰蛋白酶等）相互作用,使多酚类物质和消化酶的生物活性发生改变,进而影响多酚物质和其他营养物质的消化吸收,而RES与胃蛋白酶(Pepsin, PEP)的分子间相互作用机制未见报道。采用荧光光谱、紫外-可见吸收光谱、红外光谱和分子对接模拟等技术研究不同温度下RES与PEP相互作用的结合特性,为阐明RES和PEP的作用机制提供重要信息,同时为RES在食品和药品领域的应用提供理论参考。荧光光谱实验结果表明,PEP的荧光强度随着RES浓度的增加呈现出有规律的降低,表明RES对PEP有荧光猝灭作用。加入RES前后,PEP的紫外吸收光谱发生明显变化,初步判断RES与PEP的相互作用属于静态荧光猝灭类型;根据Stern-Volmer方程计算得到不同温度下最小猝灭速率常数K_q值远大于猝灭剂对生物大分子的最大扩散碰撞常数2.0×1010 L·mol-1·s-1,且猝灭常数(K_SV)与温度呈负相关关系,进一步验证了RES与PEP静态荧光猝灭类型结论。化学计量结合的值数目大约等于1,表明一个RES分子只能结合一个PEP分子。根据Van’t Hoff 方程以及热力学方程计算得到结果显示,ΔG＜0,说明RES与PEP的结合过程可以自发进行;ΔH＜0和ΔS＜0,表明RES与PEP之间结合作用力类型主要是氢键和范德华力。RES与PEP相互作用的同步荧光光谱和三维荧光光谱图表明,在RES的作用下,PEP的构象和微环境发生变化,色氨酸或酪氨酸残基所处微环境极性增强,疏水性减弱,蛋白构象变得疏松。红外光谱显示RES能使PEP的二级结构中α螺旋含量降低,β折叠含量增加,β转角和无规则卷曲变化不明显,这可能会影响PEP的活性。分子对接模拟实验结果显示RES与PEP中的残基Asp-32,Gly-34,Ser-35,Asn-37,Tyr-75,Gly-76,Thr-77,Ile-128,Ala-130及Gly-217有范德华力作用,与残基Ile-128及Asp-215产生超共轭效应,与残基Ser-36,Asn-37,Ile-128及Thr-218形成氢键,各种作用力使RES与PEP形成较稳定的复合物。     

多光谱法与分子对接法研究盐酸四环素与牛血清白蛋白的相互作用

盐酸四环素属于抗生素类, 目前有关盐酸四环素和牛血清白蛋白二级结构的影响及作用机理报道较少。在模拟生理条件下,采用荧光光谱法、三维荧光光谱法、紫外-可见光谱法、圆二色谱法和傅里叶红外光谱法以及分子对接模拟法,研究了盐酸四环素与牛血清白蛋白(BSA)之间的相互作用。荧光光谱表明,盐酸四环素能有效猝灭BSA的内源荧光,猝灭机制属静态猝灭,通过Stern-Volmer方程计算结合常数Ka为2.813×105 L·mol-1(298 K)。根据Vant’s Hoff方程确定结合过程中的热力学参数ΔS=-151.1 J·mol-1·K-1、ΔH=-76.09 kJ·mol-1_, 两者之间作用为氢键和范德华力。同步荧光光谱、紫外光谱、三维荧光光谱、红外光谱、圆二色谱结果证明盐酸四环素能够改变BSA的二级结构和微环境。根据Föster’s非辐射能量转移理论,盐酸四环素与BSA结合距离为0.49 nm。希尔系数(n_H)值小于1,表明盐酸四环素与BSA结合后存在药物间协同作用。圆二色谱(CD)定量测定了盐酸四环素与BSA作用前后的二级结构含量：α-螺旋含量增加了9.16%(1:1)。分子对接模拟表明盐酸四环素通过氢键、疏水作用和范德华力等多种作用力结合在BSA的site Ⅰ(亚域ⅡA)。本研究有助于了解盐酸四环素与BSA的作用机制,也有助于理解盐酸四环素对蛋白质在储运过程中功能的影响。     

Spectroscopy and Molecular Docking Study on the Interaction Behavior Between Nobiletin and Pepsin

In this study, the binding mode of nobiletin (NOB) with pepsin was investigated by spectroscopic and molecular docking methods. NOB can interact with pepsin to form a NOB-pepsin complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that NOB could spontaneously bind with pepsin through hydrophobic and electrostatic forces with one binding site. Molecular docking results revealed that NOB bound into the pepsin cavity. Synchronous and three-dimensional fluorescence spectra results provide data concerning conformational and some micro-environmental changes of pepsin. Furthermore, the binding of NOB can inhibit pepsin activity in vitro. The present study provides direct evidence at a molecular level to show that NOB could induce changes in the enzyme pepsin structure and function.     

光谱法和分子对接法研究盐酸氨溴索与人血清白蛋白的相互作用

在模拟生理条件下,用光谱法和分子对接技术研究了盐酸氨溴索与人血清白蛋白的相互作用.不同温度下的Steen-Volmer曲线和紫外-可见吸收光谱表明:盐酸氨溴索主要以动态猝灭方式使人血清白蛋白的荧光猝灭.由热力学数据确定了两者的主要作用力类型为疏水作用力.根据F(o)rster非辐射能量转移理论,得到给体-受体间的作用距...     

Study on the effect of protein on drug efficacy: cefonicid sodium–pepsin binding mechanism

The mechanism of interaction between cefonicid sodium and pepsin was investigated by various spectroscopic methods and molecular docking. Cefoncid sodium quenched the intrinsic fluorescence of pepsin at pH of 2.0 to form a new complex in a 1:1 binding mode driven by Van der Waals and hydrogen bonds. The mechanism of quenching was static. The results of molecular docking indicated that the cefonicid sodium-binding site was located in the active site of pepsin. The protein binding rates of cefonicid sodium in gastric juice was calculated and the binding model was established. It is concluded that cefonicid sodium is not suitable for oral administration.     

Investigation on the interaction of glipizide with bovine hemoglobin by spectroscopy and molecular docking

This study aims to investigate the interaction between glipizide and bovine hemoglobin using fluorescence quenching, circular dichroism spectroscopy in various temperatures (293, 303, and 310?K) and molecular docking methods. The results demonstrated that glipizide could cause strong fluorescence quenching of bovine hemoglobin by a dynamic quenching mechanism, during which the hydrophobic interaction played a dominant role in this system. The order of magnitude of binding constant is 10⁴, and the number of binding site in the system was close to 1. It also showed that tyrosine residues and tryptophan residues were both involved in the binding of glipizide with bovine hemoglobin, and was closer to the later. Circular dichroism spectra revealed that the conformation of bovine hemoglobin was changed during the binding reaction. The interaction of the system was studied by both spectroscopic method and molecular docking simulation, and the conclusions are consistent.     

Interaction of quercetin with ovalbumin: Spectroscopic and molecular modeling studies

The binding of quercetin (QCT) to ovalbumin (OVA) in aqueous solution was investigated by molecular spectroscopy and modeling at pH 7.4. The fluorescence, synchronous fluorescence and UV-absorption spectroscopies were employed to study the mode and the mechanism for this interaction. QCT binding is characterized by one high affinity binding site with the association constants of the order of 10⁵. The distance between donor (OVA) and acceptor (QCT) was estimated according to Forster's theory of non-radiation energy transfer. Molecular docking showed that the QCT can bind to the active site of OVA. The binding dynamics was expounded by thermodynamic parameters, molecular modeling and accessible surface area calculation, which entails that hydrophobic interactions, hydrogen bonding and electrostatic forces stabilizes the interaction.     

Interaction of human serum albumin with N-(4-ethoxyphenyl)-N′-(4-antipyrinyl) thiourea using spectroscopies and molecular modeling method

The interaction between N-(4-ethoxyphenyl)-N′-(4-antipyrinyl) thiourea (EPAT) and human serum albumin (HSA) was studied by fluorescence spectroscopy in combination with UV absorption spectroscopy. The intrinsic fluorescence of human serum albumin was quenched by EPAT through a static quenching procedure. The binding constants of EPAT with HSA were estimated according to the fluorescence quenching results at different temperatures. The binding distance was obtained and the binding force was suggested to be mainly hydrophobic force, which was in accordance with the study of molecular model. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented, and results were very satisfactory.     

光谱法结合分子模拟技术研究左氧氟沙星与胃蛋白酶的相互作用机理

在模拟生理条件下,采用荧光光谱法结合分子对接技术研究了左氧氟沙星与胃蛋白酶的相互作用。不同温度下的Stern-Volmer曲线结果表明,左氧氟沙星主要以静态猝灭的方式使胃蛋白酶的荧光产生猝灭。由热力学数据确定了两者之间存在的作用力主要为静电作用力。三维荧光光谱实验结果表明,在左氧氟沙星与胃蛋白酶发生作用过程中引起了胃蛋白酶中酪氨酸和色氨酸残基构象的变化,从而导致胃蛋白酶发生了猝灭作用。为了进一步探讨左氧氟沙星与胃蛋白酶相互作用的分子机理,利用分子对接技术对两者的相互作用进行模拟。结果表明两者发生作用的区域位于胃蛋白酶的催化活性中心,左氧氟沙星的进入导致酶催化中心氨基酸残基的微环境发生改变,从而对胃蛋白酶的活性造成影响,这可能也是左氧氟沙星引起消化不良的主要原因。     

多光谱法和分子对接模拟法研究黄腐植酸和牛血清白蛋白的相互作用

在模拟生理环境中,使用荧光光谱法、紫外光谱法、圆二色谱法、同步荧光光谱法、三维荧光光谱法与分子对接模拟法研究黄腐植酸和牛血清白蛋白(BSA)之间相互作用。在荧光光谱法研究中,经Stern-Volmer方程计算得到298,303和308 K温度下的动态荧光猝灭速率常数K_q和猝灭常数,证明BSA与黄腐殖酸(FA)相互作用的猝灭过程为静态猝灭;同时根据计算得出的结合位点数n都在1附近,FA与BSA体系相互作用比为1∶1;利用静态猝灭双对数方程计算三个温度下的热力学参数,焓变ΔH<0,熵变ΔS＜0,得出结论,FA与BSA之间的主要作用力为氢键和范德华力;ΔG＜0,说明作用过程为自发过程。采用Förster’s偶极-偶极非辐射能量转移理论,计算出结合距离r=6.340 nm,表明BSA与FA之间存在非辐射能量转移。分子对接模拟结果表明FA与BSA残基的结合作用力具有氢键和范德华力,同时二者之间还存在疏水作用力,多种力共同作用使FA与BSA能够稳定结合。通过对FA与BSA相互作用的紫外-可见吸收光谱分析,发现BSA最大吸收峰发生了较为明显的红移,表明FA使BSA的二级结构发生改变。通过研究FA与BSA相互作用的同步荧光光谱,得到FA使BSA中的色氨酸（Trp）残基周围的微环境极性增强,疏水性减弱,亲水性增强,使BSA的蛋白质构象发生了一定程度的改变。通过研究FA与BSA相互作用的三维荧光光谱,峰1（peak 1）与峰2（peak 2）的最大发射波长峰都发生了红移,证明FA与BSA发生了相互作用,FA使BSA周围环境的极性增大,疏水性减小,亲水性增加,BSA蛋白质构象发生变化。最后采用圆二色谱法进行分析,利用软件计算得出该实验相互作用体系下α-螺旋（α-Helix）减少2.3％、β-折叠（β-sheet）增加7.7％、β-转角（β-Turn）增加0.6％和无规则结构（Random coil）含量减少1.2％,β-折叠（β-sheet）含量增加最为明显, 强有力地说明了FA使BSA结构发生了改变。     

两种肉桂酸肟酯衍生物的合成及其与人血清白蛋白的结合

以间甲氧基肉桂酸、对位取代的苯甲醛为原料,设计合成了2种未见报道的肉桂酸肟酯类衍生物,并用MS、IR、1H NMR、13C NMR进行结构表征。采用分子对接技术和荧光光谱法、紫外-可见光谱法、位点竞争法研究了2种衍生物分别和人血清白蛋白(HSA)相结合的机理。通过Stern-Volmer方程等处理荧光猝灭相关数据得到了衍生物与HSA相互作用的结合常数和热力学参数。结合紫外-可见光谱对两种衍生物与HSA的相互作用进行了进一步的分析,结果表明在体外生理条件下,衍生物都可以与HSA结合,对HSA内源荧光产生静态猝灭并对其构象产生影响,其主要的结合力为氢键和范德华力。位点竞争实验表明衍生物与HSA相互作用都发生在Sudlow site 1(亚域ⅡA)处。以上实验结果均验证了分子模拟对实验的预测。     

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.     

贝诺酯与牛血清白蛋白位点选择性结合的分子光谱研究

采用荧光光谱、紫外可见光谱、同步荧光光谱及三维荧光光谱等分子光谱方法,研究了生理条件下贝诺酯(BEN)与牛血清白蛋白(BSA)的相互作用。结果表明,BEN对BSA的内源荧光有显著的猝灭作用,猝灭机理为动态猝灭,二者之间的作用力类型以疏水作用为主,BEN与BSA发生反应后,使BSA的疏水环境极性增强,疏水性减弱,荧光强度降低。测得的表观结合常数和结合位点数分别是1 050 L·mol^-1和0.88,同时测得了焓变(ΔH)、熵变(ΔS)和自由能变(ΔG)等热力学参数。同步荧光和三维荧光光谱的结果表明,BEN使BSA的构象发生改变。利用荧光特异性位点探针DA和DP,通过竞争结合实验,监测BEN与BSA的结合位点,测得了位点Ⅰ和位点Ⅱ的表观结合常数分别为4 300 L·mol^-1和21 200 L·mol^-1,表明BEN与BSA优先在位点Ⅱ结合。     

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.     

Interaction mechanism of 2-aminobenzothiazole with herring sperm DNA

The toxic interaction of 2-aminobenzothiazole (2-ABT) with herring sperm DNA (hs-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated that 2-ABT interacted with hs-DNA in a minor groove binding mode. The binding constant and the number of binding sites were 7.2×10³ L mol^?1 and 0.95, respectively. Circular dichroism spectroscopy (CD) was employed to measure the conformation change of hs-DNA in the presence of 2-ABT, which verified the minor groove binding mode. The molecular modeling results illustrated that 2-ABT tended to bind in the region of rich A–T base pairs through the hydrogen bond between A 18 and amino group of 2-ABT. Sequence specificity was confirmed by comparison on the interactions of 2-ABT with four kinds of bases. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with biomacromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.     

胡椒碱与牛血清白蛋白的相互作用

采用荧光光谱技术研究了胡椒碱与牛血清白蛋白（BSA）的相互作用。根据测定不同温度下胡椒碱对BSA的猝灭常数,证实了荧光猝灭过程为静态猝灭,由热力学参数焓变（ΔH）小于零和熵变（ΔS）大于零,推断出胡椒碱与BSA之间主要靠静电引力相结合,生成自由能变（ΔG）为负值,表明胡椒碱与BSA的作用过程是一个自发过程;并应用同步荧光光谱技术考察了胡椒碱对BSA构象的影响。     

Photophysics of 1-ethynylpyrene-modified RNA base adenine

The photophysical behaviour of a pyrene-modified adenine base is examined. Time-dependent absorption and fluorescence spectroscopy is used to shed light on the fluorescence behavior and the charge transfer between the fluorophore and the nucleobase. The results from TCSPC, fluorescence upconversion and pump-probe absorption spectroscopy are reported, compared and analyzed. This study is a prerequisite for the detailed characterization of RNA dynamics in real time and on a molecular time scale.     

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.