Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic,calorimetric and modeling investigations

One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10⁴ M^?1. According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven.     

Biophysical Studies on the Interactions of a Classic Mitochondrial Uncoupler with Bovine Serum Albumin by Spectroscopic,Isothermal Titration Calorimetric and Molecular Modeling Methods

The interaction between a classic uncoupler (2,4-dinitrophenol, DNP) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy under the physiological conditions. The fluorescence quenching constants were calculated by the Stern-Volmer equation, and based upon the temperature dependence of quenching constants, it was proved that DNP caused a static quenching of the intrinsic fluorescence of BSA. Owing to the static quenching mechanism, different associative binding constants at various temperatures were determined and thus the thermodynamic parameters, namely enthalpy (ΔH = −21.12 kJ mol⁻¹) and entropy changes (ΔS = 23.51 J mol⁻¹ K⁻¹) could be calculated based on the binding constants. Moreover, the enthalpy and entropy changes are consistent with the “Enthalpy-Entropy Compensation” equation obtained from our previous work. The negative enthalpy and positive entropy indicated that the electrostatic interactions played a major role in DNP-BSA binding process. Site marker competitive displacement experiments were carried out by using fluorescence and isothermal titration calorimetry (ITC) methods. These results showed that DNP bound with high affinity to Sudlow’s site I (subdomain IIA) of BSA. The distance (r = 3.78 nm) between donor (BSA) and acceptor (DNP) was obtained according to the mechanism of fluorescence resonance energy transfer (FRET). Furthermore, the results of synchronous fluorescence and circular dichroism (CD) spectroscopic studies indicated that the microenvironment and the secondary conformation of BSA were altered. The above results were supported by theoretical molecular modeling methods.     

ITC研究达卡巴嗪与DNA反应动力学

利用等温滴定量热(ITC)、光谱、粘度测量等方法,研究了小牛胸腺DNA与抗癌药物达卡巴嗪的相互作用。结果表明:达卡巴嗪与DNA作用后,吸收光谱会出现增色、蓝移和粘度减小等现象。采用ITC法得到了结合常数以及结合位点数,发现达卡巴嗪与DNA以非经典嵌插式及表面作用两种方式结合。对于嵌插式,ΔH10,ΔS10,K1=5.63×10~4,结合位点数0.10;对于药物分子仅与DNA表面发生作用而并未嵌入到DNA分子的疏水部分的结合方式,ΔH_20,ΔS_20,K_2=1.00×10~3,结合位点数9.99。同时发现紫外法得到的结合常数是Ka=6.70×10~4,与ITC的嵌插式吻合。     

光谱法研究普利沙星与小牛胸腺DNA的结合作用

利用紫外光谱,荧光光谱及流体动力学方法,研究了普利沙星与小牛胸腺DNA的作用机理。讨论了不同浓度普利沙星与DNA作用的紫外光谱,荧光光谱,磷酸盐效应以及离子强度对两者相互作用的影响,测量了DNA的热变性温度和粘度。从紫外光谱图上看出DNA发生了明显的减色效应, 说明普利沙星可能与DNA发生作用。普利沙星的荧光光谱发生了有规律的猝灭,最大发射峰发生红移, 猝灭常数为3.1×104 L·mol-1, 为静态猝灭,表明普利沙星与DNA结合生成了二元复合物。磷酸盐效应表明普利沙星与DNA的磷酸基团不发生静电作用。普利沙星引起了DNA的热变性温度略微升高(≤7℃)和DNA粘度略微下降, 表明普利沙星与DNA之间不存在插入作用, 只是在DNA的外部发生沟槽作用。     

低聚壳聚糖衍生物与BSA的作用研究

研究了具有生物相容性的低聚壳聚糖衍生物与活性药物牛血清白蛋白(BSA)之间的作用。合成了聚合度为20的窄分子量分布低聚壳聚糖 (NLCS₂₀)的3种衍生物：N-马来酰化低聚壳聚糖(MNLCS)、N-羧甲基低聚壳聚糖(CNLCS)和N-琥珀酰化低聚壳聚糖(SNLCS)。通过紫外光谱(UV)、荧光光谱和等温滴定微量热仪(ITC)研究了NLCS₂₀衍生物与BSA之间的相互作用。结果表明,BSA的构象在NLCS₂₀衍生物溶液微环境中没有发生变化,活性也没有受到影响。NLCS₂₀衍生物与BSA的作用过程是放热的且是自发进行的,主要作用力是氢键和疏水作用。说明低聚壳聚糖衍生物能够作为潜在的亲水药物如活性医药蛋白或氨基酸的运输载体。     

Comprehensive Study on the Binding of Iron Schiff Base Complex with DNA and Determining the Binding Mode

The iron (III) [N, N′ Bis (5-(triphenyl phosphonium methyl) salicylidene)-1, 2 ethanediamine] chloride [Fe Salen]Cl, has been synthesized and characterized as described previously. The interaction of iron complex with calf thymus (CT) DNA has been studied extensively by experimental techniques. Absorption spectra showed both hypochromism and hyperchromism. Thermal denaturation study of DNA with complex revealed the ΔT_m of 5 °C. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by adding of the iron complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be 5?×?10⁶?μM^?1. Fluorescence Scatchard plot revealed type B behavior for interaction of complex to DNA. Circular dichroism (CD) spectra measurements showed that the complex interacts with DNA via surface and groove bindings. Linear dichroism (LD) measurements confirmed the bending of DNA in the presence of complex. Furthermore, Isothermal titration calorimetry (ITC) experiments approved that the binding of complex is based on both electrostatic and hydrophobic interactions. More, ITC profile exhibits the existence of two binding phases for the complex.     

光谱及等温滴定量热法探究盐酸阿霉素与DNA作用机理

利用荧光光谱法,紫外可见光谱法,红外光谱法,圆二色谱法和等温滴定量热法等手段,对抗癌药物盐酸阿霉素 (DOX) 与DNA的作用过程进行研究,测得了它们的结合常数K_a、结合位点数n、反应焓变ΔH、熵变ΔS及ΔG,且在结合过程中,B型DNA的螺旋结构在一定程度发生改变。荧光光谱的数据显示出显著的猝灭效应, 表明DNA是一个DOX荧光的很好的猝灭剂。红外光谱表明阳离子DOX+通过静电吸引与 DNA 的磷酸基团相互作用,且DOX的碳氢链通过疏水缔合与DNA作用。ITC测定了DOX和DNA相互作用的焓变和熵变,表明DOX的烃链和DNA的碱基之间的疏水性相互作用提供了结合的驱动力。     

Binding Studies of Isoxsuprine Hydrochloride to Calf Thymus DNA Using Multispectroscopic and Molecular Docking Techniques

In the present work, the interaction of Isoxsuprine (ISX) with Calf thymus DNA (ct-DNA) under physiological conditions (Tris–HCl buffer of pH 7.4) was investigated by using electronic absorption, circular dichroism, viscosity, electrochemical studies, fluorescence techniques, salt effect studies and computational studies. Competitive fluorimetric studies with Hoechst 33258 have shown that ISX exhibit the ability to displace the DNA-bound Hoechst 33258, indicating that it binds to ct-DNA in strong competition with Hoechst 33258 for the minor groove binding. Furthermore, the resulting data showed that ISX cannot displace methylene blue or acridine orange, which are the common intercalator molecules. The viscosity of ct-DNA solution was almost unchanged on addition of ISX and circular dichroism (CD) spectra of ct-DNA showed small changes in the presence of ISX which is in agreement with groove binding mode of interaction. Thus all above studies showed that the ISX drug binds to ct-DNA in a groove binding mode.The salt-effect studies showed the non-electrostatic nature of binding of ISX to ct-DNA. Moreover, molecular docking results support the above experimental data and suggest that ISX prefers to bind on the minor groove of ct-DNA.     

血管紧张素转化酶抑制肽Leu-Lys-Pro（LKP）抑制机理的研究

血管紧张素转化酶（ACE）是一种含锌离子的羧二肽酶,通过肾素-血管紧张素系统和激肽释放酶-激肽系统进行血压调节。食源血管紧张素转化酶抑制肽（ACEIP）可抑制ACE的活性对高血压控制有利。以鲣鱼蛋白分离出的ACE抑制肽Leu-Lys-Pro（LKP）为原料,采用荧光光谱法、紫外-可见光谱法、圆二色谱法、等温滴定量热法（ITC）以及分子对接技术研究了LKP对ACE的抑制机理。荧光光谱结果表明,LKP能够有效猝灭ACE的内源荧光,猝灭机制为静态猝灭,两者结合可形成较稳定的复合物,ACE中色氨酸和酪氨酸残基所处的微环境疏水性减小,导致极性增强。紫外、圆二色谱结果表明,LKP与ACE结合会导致ACE构象发生改变,ACE与LKP结合后二级结构比未结合时松散,为紧密-松散-稍紧密的变化过程。ITC测得LKP与ACE结合的焓变（ΔH）、熵变（ΔS）、化学计量比（n）以及结合常数（K_a）等热力学参数,结果表明两者结合反应是由熵驱动的自发吸热过程,结合力主要为疏水作用,确定LKP与ACE相互作用的结合位点数约为1,且随温度升高而增加。LKP与ACE的结合常数K_a分别为2.2×103,0.9×103和5.3×103,说明两者亲和力较小。分子对接结果表明,ACE活性中心的S₁口袋的氨基酸残基Gln281,Lys511与LKP形成氢键相互作用,His353,His513与LKP具有疏水作用,LKP主要通过疏水作用与ACE结合,氢键稳定蛋白空间结构。该研究对了解ACE抑制肽与ACE的作用机制提供了一定的帮助,为开发新的治疗高血压药物建立一定的理论基础。     

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.     

光谱法联合分子对接和等温滴定微量热法研究全氟壬酸与人血清白蛋白的相互作用

全氟壬酸(PFNA)是在血清中检测到第三多的全氟烷酸类(PFAAs)新型有毒环境污染物。目前PFNA对人血清白蛋白(HSA)结构甚至是功能的影响还处于起步阶段,借助于多光谱、分子对接和等温滴定微量热(ITC)技术研究了PFNA和HSA相互作用的结合机理。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。荧光结果表明PFNA通过动静态猝灭方式可以猝灭HSA的内源荧光。取代实验和分子对接结果表明,PFNA主要通过极性键、疏水力和卤素键键合在HSA亚域ⅡA疏水腔中,最佳对接自由能为-26.54 kJ·mol-1,表明PFNA分子与HSA有较大的结合亲和力。ITC表明两者的结合属于两类结合位点模型并给出了相应的热力学参数：第一类结合位点有较大的亲和力,属于焓驱动,静电力和卤键作为主要驱动力;第二类结合位点亲和力较小,主要驱动力是疏水力。三维荧光光谱揭示PFNA与HSA生成复合物后,可以改变HSA的构象,引起Trp和Tyr残基微环境疏水性增强。圆二色谱(CD)定量测定了HSA与PFNA作用前后的二级结构含量：α-螺旋、β-折叠和β-转角含量分别降低14.3%,5.3%和3.5%,无规卷曲含量从14.4%增加到37.5%。以上结果表明,PFNA与HSA的结合可以改变HSA的二级结构,进而可能影响HSA的生理功能。结果阐述了PFNA与HSA相互作用机理,并且为PFNA在体内的运输和分配提供了可靠的生物物理和生物化学的相关依据。     

溴鼠灵与DNA作用机制的光谱研究

农用化学物质残留可能会与DNA结合形成加和物,从而对动物和人类造成危害。文章利用紫外光谱和荧光光谱研究了溴鼠灵(BDF)与小牛胸腺DNA(ct-DNA)的作用机理。结果表明,低浓度的ct-DNA可使BDF的吸收光谱发生减色,ct-DNA对BDF的稳态荧光有明显的的猝灭作用,属于静态猝灭方式,27 ℃时K_SV=1.21×104 L·mol-1;根据热力学参数推测BDF与ct-DNA之间的相互作用主要是范德瓦耳斯力作用;猝灭试验发现ct-DNA对BDF无明显保护作用;离子强度改变时,BDF/ct-DNA体系的荧光强度也随之改变;ct-DNA对β-CD/BDF包络物的荧光也有猝灭,但猝灭程度比游离的BDF有所降低,表明β-CD对BDF具有保护作用,也说明BDF与ct-DNA或β-CD的结合具有竞争性。各试验结果一致表明BDF与ct-DNA之间主要是以沟区结合方式发生作用。另外,试验结果也表明BDF与ct-DNA之间还存在静电作用。范德瓦耳斯力和静电作用的共同作用,为BDF在沟区与DNA的相互结合提供了更好条件。     

金属离子诱导下DNA折叠的不同机理

利用等温滴定量热仪、圆二色谱和荧光光谱,研究和分析了人工合成的DNA单链序列d(T₆C₆T₆C₆T₆C₆T₆)与Hg²⁺和Ag⁺相互作用的折叠过程. 在改变离子添加顺序的情况下,尽管热力学数据显示两种情况下都能通过两种不同反应路径得到一种相对稳定的发夹结构,但等温滴定量热仪数据却显示最终产物形成的机理截然不同.当先加入Hg²⁺时,发夹结构首先通过T-Hg-T碱基对形成然后C-Ag-C碱基对得到进一步稳定. 然而当先加入Ag⁺时,通过圆二色谱和荧光分析确认了一种不常见的金属碱基对T-Ag-C取代了经典的C-Ag-C碱基对.     

Study on the Interaction of an Anthracycline Disaccharide with DNA by Spectroscopic Techniques and Molecular Modeling

This study was designed to examine the interaction of 4'-O-(a-L-Cladinosyl) daunorubicin (DNR-D5), a disaccharide anthracycline with calf thymus deoxyribonucleic acid (ctDNA) by UV/Vis in combination with fluorescence spectroscopy and molecular modeling techniques under physiological conditions (Britton-Robinson buffer solutions, pH?=?7.4). By the analysis of UV/Vis spectrum, it was observed that upon binding to ctDNA the anthraquinone chromophore of DNR-D5 could slide into the base pairs. Moreover, the large binding constant indicated DNR-D5 had a high affinity with ctDNA. At the same time, fluorescence spectra suggested that the quenching mechanism of the interaction of DNR-D5 to ctDNA was a static quenching type. The binding constants between DNR-D5 and ctDNA were calculated based on fluorescence quenching data at different temperatures. The negative ?G implied that the binding process was spontaneous, and negative ?H and negative ΔS suggested that hydrogen bonding force most likely played a major role in the binding of DNR-D5 to ctDNA. Moreover, the results obtained from molecular docking corroborate the experimental results obtained from spectroscopic investigations.     

光谱法研究Leu-ENK与ct-DNA的相互作用

亮氨酸脑啡五肽是动物体内一种具有很强生物活性的阿片肽,在生物体内有极其重要的作用.利用紫外光谱和荧光光谱法研究了亮氨酸脑啡五肽(Leu-ENK)与小牛胸腺DNA(ct-DNA)的相互作用.研究表明,Leu-ENK-ct-DNA体系随着Leu-ENK浓度增大,体系的紫外光谱呈增色效应；随着ct-DNA浓度的增加,体系Le...     

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.     

光谱法研究山奈酚与ct-DNA的相互作用

利用紫外-可见吸收光谱和荧光光谱研究了黄酮类化合物山奈酚与小牛胸腺DNA（ct-DNA）之间的相互作用。随着DNA的加入,山奈酚的吸收光谱逐渐发生明显的红移和减色效应,通过双倒数法拟合分析,得到山奈酚与ct-DNA的结合常数为1.2×10^4。山奈酚自身荧光很弱,与DNA结合后其荧光明显增强,表明山奈酚可以作为荧光探针来开展与生物大分子相互作用的研究。     

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.     

Thionine Interaction to DNA: Comparative Spectroscopic Studies on Double Stranded Versus Single Stranded DNA

Interaction of thionine with double stranded and single stranded calf thymus DNA has been studied by absorbance, fluorescence, competition dialysis, circular dichroism and isothermal titration calorimetry. Binding to the native double stranded DNA conformation induced strong quenching in fluorescence spectrum of thionine. Linear Scatchard plots indicated the binding to be of one type and the affinity values evaluated to be of the order of 10⁵ M⁻¹ with double stranded DNA. Fluorescence quenching was much weaker with single stranded DNA and the binding affinity was about one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of dye molecules bound to the double stranded DNA was quenched much less compared to those bound to the single stranded DNA. Furthermore, there was significant emission polarization for the bound dye molecules and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding to ds DNA. Salt dependence of the binding phenomenon revealed that electrostatic forces played a significant role in the binding process. The intercalation of the dye molecules to double stranded DNA and simple stacking to single strands was proved from these fluorescence techniques. Support to the fluorescence results have been derived from absorption, circular dichroic and dialysis results. Calorimetric studies suggested that the binding to ds DNA conformation was both enthalpy and entropy favoured while that to ss DNA was predominantly entropy favoured.     

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.