光谱法研究柔红霉素衍生物DNR-D3与DNA相互作用

在人体生理条件下(pH7.4),利用紫外光谱法和荧光光谱法研究了本实验室合成的柔红霉素衍生物DNR-D3与小牛胸腺DNA(ctDNA)的相互作用。实验发现,在ctDNA存在下DNR-D3的紫外吸收光谱发生减色效应且出现红移现象,这表明DNR-D3与ctDNA相互作用的结合方式以嵌插为主。通过20,30,37℃条件下ctDNA与DNR-D3相互作用的荧光光谱,判断ctDNA与DNR-D3之间荧光猝灭方式为静态猝灭。利用荧光数据计算不同温度下的结合常数,结合位点数及热力学参数,从而判断DNR-D3与ctDNA的作用方式以嵌插为主,作用力类型是以氢键和静电作用为主,此过程是放热的焓熵协同驱动过程。DNR-D3的荧光猝灭50%时DNR-D3与ctDNA的摩尔浓度比Rc=7/25,这表明DNR-D3的蒽环与ctDNA发生了强烈的嵌插作用,DNR-D3显示出了较强的抗癌活性。通过研究可知,DNR-D3有望成为抗癌活性候选药物。     

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.     

Spectroscopic,Viscositic and Molecular Modeling Studies on the Interaction of 3′-Azido-Daunorubicin Thiosemicarbazone with DNA

A new daunorubicin has been synthesized and structurally characterized. The interaction of native calf thymus DNA (ctDNA) with 3′-azido-daunorubicin thiosemicarbazone (ADNRT) was investigated under simulated physiological conditions by multi-spectroscopic techniques, viscometric measurements and molecular modeling study. It concluded that ADNRT could intercalate into the base pairs of ctDNA, and the fluorescence quenching by ctDNA was static quenching type. Thermodynamic parameters calculated suggested that the binding of ADNRT to ctDNA was mainly driven by hydrophobic interactions. The relative viscosity of ctDNA increased with the addition of ADNRT, which confirmed the intercalation mode. Furthermore, molecular modeling studies corroborate the above experimental results.     

重金属Cu2+增强农药甲萘威对ctDNA作用的光谱法研究

运用荧光光谱和紫外吸收光谱研究了在Cu2 的参与下农药甲萘威与小牛胸腺DNA(ctDNA)的相互作用.实验结果表明,甲萘威可以嵌入小牛胸腺DNA的双链中形成DNA加合物,从而使甲萘威的荧光光谱发生猝灭.通过计算这种猝灭为静态猝灭.当有Cu2 的参与下,甲萘威的猝灭常数增大,结合位点数n也有很大的增加.通过对热力学函数的计算和分析,在有Cu2 参与时,金属离子可能在甲萘威分子与ctD-NA分子间起"离子架桥作用",使甲萘威分子与ctDNA分子间静电相互作用增强,故△H对△G的贡献增大.     

环丙氟沙星荧光探针对小牛胸腺DNA相互作用的研究

使用紫外和荧光光谱法研究了环丙氟沙星(CIF)和小牛胸腺DNA(ctDNA)之间的相互作用。在pH 7.0磷酸盐缓冲液中,CIF在270 nm激发和在420 nm处有很强的荧光发射。它们间的沟槽作用导致小牛胸腺ctDNA对CIF的荧光存在着很强的猝灭作用,猝灭常数为2.64×104 mol-1L(25 ℃)。利用ctDNA对CIF的荧光猝灭作用建立了核酸测定的新方法,线性范围为80 nmol·L-1～45 μmol·L-1,对25 μmol·L-1ctDNA的相对标准偏差为4.2％。     

Interaction Mechanism of Trp-Arg Dipeptide with Calf Thymus DNA

The interaction between Trp-Arg dipetide (WR) and calf thymus DNA (ctDNA) in pH 7.4 Tris-HCl buffer was investigated by multi-spectroscopic techniques and molecular modeling. The fluorescence spectroscopy and UV absorption spectroscopy indicated that WR interacted with ctDNA in a minor groove binding mode and the binding constant was 4.1?×?10³. The ionic strength effect and single-stranded DNA (ssDNA) quenching effect further verified the minor groove binding mode. Circular dichroism spectroscopy (CD) was employed to measure the conformation change of ctDNA in the presence of WR. The molecular modeling results illustrated that electrostatic interaction and groove binding coexisted between them and the hydrogen bond and Van der Waals were main acting forces. All the above methods can be widely used to investigate the interaction of peptide with nucleic acids, which contributes to design the structure of new and efficient drugs.

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Fōrster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4.05 nm; 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。     

Interaction Between Methylene Blue and CalfThymus Deoxyribonucleic Acid by Spectroscopic Technologies

Characterization of the interaction between methylene blue (MB) and calf thymus deoxyribonucleic acid (ctDNA) was investigated by UV absorption spectra, fluorescence spectra, fluorescence polarization and fluorescence quenching experiments by ferrocyanide. The above results indicated that the binding modes of MB to ctDNA were relative to the molar ratio γ (γ=[DNA]/[MB]). At low γ ratios (γ < 4), remarkable hypochromic effect with no shift of λ_max in the absorption spectra of MB was observed in the presence of increasing amounts of ctDNA, the fluorescence of MB was efficiently quenched by the ctDNA bases and the fluorescence polarization of MB was slightly increased, which indicated that MB cations bound to phosphate groups of ctDNA by electrostatic interaction and then stacked on the surface of ctDNA helix. While at high γ ratios (γ > 6), besides the fluorescence of MB was quenched efficiently by the ctDNA bases, a red shift (about 3 nm) in the absorption spectra of MB was observed and the fluorescence polarization of MB was obviously increased, which indicated the intercalation binding that MB molecules were intercalated into the space of two neighbouring DNA base pairs was the preferred mode. Effects of K₄Fe(CN)₆ on the fluorescence quenching of the MB-ctDNA system at low and high γ ratios were also performed. The results showed that at γ = 1.7, the quenching effect by ferrocyanide was higher than that of pure MB, while at γ = 13.6 a decreased quenching of the fluorescence intensity was observed as compared with that of pure MB, which further proved the above conclusion. In addition, the mechanisms of the hypochromic effect and the fluorescence quenching were also discussed in detail.     

Interaction of Anthracycline 3′-azido-epirubicin with Calf Thymus DNA via Spectral and Molecular Modeling Techniques

Anthracycline antibiotics are extensively applied to clinical antitumor therapy. The binding mode and mechanism of a new anthracycline 3′-azido-epirubicin (AEPI) with calf thymus deoxyribonucleic acid (ctDNA) were investigated employing multiple spectroscopy techniques in Tris-HCl buffer solution (pH 7.4). Effect of pH on the interaction was provided to determine the proper environment for whole research. Iodide quenching studies and fluorescence polarization measurement indicated that ctDNA quenched the fluorescence of AEPI significantly via intercalation binding mode. The binding constants and binding sites for the interaction were calculated. From binding constant dependence on the temperature, static quenching mechanism of AEPI by ctDNA was confirmed based on the Stern-Volmer equation. Additionally, the thermodynamic parameters for the reaction revealed that the van der Waals force and hydrogen bonding were the main acting forces in the binding process. Molecular modeling result indicated that the hydrogen bonding played a major role in the binding of AEPI to ctDNA.     

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.     

桑色素与牛血红蛋白相互作用的光谱研究

利用紫外可见吸收光谱和荧光光谱研究了在生理pH条件下桑色素与牛血红蛋白(BHb)的相互作用。实验结果表明:桑色素分子与BHb发生反应生成基态复合物,导致BHb内源荧光的猝灭,该猝灭属于静态猝灭。测定了不同温度下该反应的表观结合常数、结合位点数及结合热力学参数,热力学参数的变化表明上述作用过程是一个熵增加、自由能降低的自发分子间作用过程,桑色素与BHb之间以疏水和静电作用力为主;根据F-rster能量转移理论,测得供体与受体间结合距离r和能量转移效率E;并用同步荧光光谱法探讨了桑色素对BHb构象的影响。     

Research on the interaction of cr(III) complex of genistein with DNA

The interaction of the Cr(III) complex of genistein (GEN-Cr) with calf thymus DNA (ctDNA) in Tris (pH 7.2) buffer was investigated using UV spectra, DNA melting, fluorescence spectra and viscosity. From the absorption titration experiment, no obvious red shift was found, but the notable hypochromicities were observed. When C(DNA)/C(GEN-Cr) = 3, the pi-pi* transitions of the complex at 272 nm showed a decrease in intensity of 29.1%, which indicated that there was remarkable intercalation between complex and DNA base pairs, involving a strong pi-stacking interacting between them. The binding constant for the complex was K = 1.9 x 10(5) mol x L(-1). From the melting curves of ctDNA in the absence and presence of the complex, the melting temperature of ctDNA was found to increase by 5.5 degrees C from 74 to 79.5 degrees C, owing to the increased stability of the helix in the presence of the complex that was intercalated into the double helix. The complex could emit weak luminescence in Tris buffer. The emission intensity of the complex at 340 nm increased steadily with the addition of ctDNA. The result suggested that the complex got into a hydrophobic environment inside the DNA and avoided the effect of solvent water molecules. The strong interaction of the complex and ctDNA also resulted in greatly enhanced intensity of the resonance light scattering spectra. The emission intensity of DNA-EB system at 600 nm decreased remarkably with increasing the complex concentration, which indicated that the complex could be intercalated into DNA and replace EB from the DNA-EB system. According to the classical Stern-Volmer equation, the quenching plots at 25 and 37 degrees C both appeared approximately linear. These results showed that there was one predominant quenching style in this process. Viscosity experiments were carried out by an Ubbelodhe viscometer at 20.0 (+/- 0.1) degrees C. The relative viscosity of ctDNA increased steadily with the increased in the complex. The result clearly showed that the complex could be intercalated between DNA base pairs, causing an extension of the helix, and thus increased the viscosity of DNA. The results above indicated that there is a relatively strong interaction between the GEN-Cr complex and ctDNA, and the complex could bind ctDNA mainly by intercalation. The research suggested that the GEN-Cr complex may be a promising candidate for anticancer, which deserves further research.     

Interaction of One Anthraquinone Derivative with ctDNA Analyzed by Spectroscopic and Modeling Methods

The interaction of one anthraquinone derivative (AOMan) with calf thymus deoxyribonucleic acid (ctDNA) was systematically investigated at physiological pH 7.4 by fluorescence spectroscopy and molecular modeling. Binding constants of ctDNA with AOMan were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van’t Hoff equation, which indicated that the reaction was spontaneous and predominantly enthalpically driven. The increasing viscosity of ctDNA indicated that AOMan could intercalate into the base pairs of ctDNA. This conclusion was also demonstrated by the results obtained from KI quenching, denatured DNA studies and fluorescence polarization experiment. Furthermore, the molecular modeling results showed that anthraquinone ring tended to slide into the G–C rich region of ctDNA through the hydrogen bond, which are consistent with the results from experimental methods. Studying the binding interaction of target anthraquinones with DNA is one of the key steps in their DNA-changing action and the design of new drugs.     

应用荧光猝灭法研究尼莫地平与牛血清白蛋白的相互作用

应用荧光光谱(FS)和紫外光谱(UV)研究了尼莫地平与牛血清白蛋白(BSA)之间的相互作用。尼莫地平与BSA的结合常数K_A为5.01×104(26 ℃)和4.46×104(36 ℃),尼莫地平在BSA上的结合位点数为1.08±0.01。根据Frster非辐射能量转移理论,求出了尼莫地平与BSA之间的结合距离为3.14 nm(26 ℃)和3.10 nm(36 ℃)。实验表明静态猝灭和非辐射能量转移是 导致尼莫地平对BSA荧光猝灭的两大原因。通过计算热力学参数,可知该药物与牛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,且二者之间的作用力以静电相互作用为主。     

Fluorimetric Study on the Interaction between Norfloxacin and Proflavine Hemisulphate

The interaction between Norfloxacin (NF) and Proflavine hemisulphate (PF) was investigated by spectroscopic tools like UV–VIS absorption and Fluorescence spectroscopy. It was proved that fluorescence quenching of NF by PF is due to the formation of NF-PF complex which was supported by UV–VIS absorption study. The study of thermodynamic parameters suggested that the key interacting forces are hydrogen bond and van der Waal’s interactions and the binding interaction was spontaneous. The distance r between NF and PF was obtained according to the Förster’s theory of non-radiative energy transfer. The fluorescence quenching mechanism was applied to estimate PF directly from pharmaceutical samples.     

荧光光谱法研究拜复乐与小牛胸腺DNA的作用机理

用荧光光谱法研究了拜复乐（MXFX）与小牛胸腺DNA（ctDNA）之间的相互作用。在pH=7.4的Tris-HCl缓冲溶液中,MXFX的荧光激发峰和发射峰分别位于291 nm和462 nm。ctDNA的加入对MXFX的荧光有静态猝灭作用,这种荧光静态猝灭作用是由ctDNA和MXFX结合引起的,作用力为氢键或范德华力,结合常数为1.28×10⁵ L/mol（25 ℃）。采用离子强度的影响、碘离子猝灭及溴乙锭竞争作用实验研究了MXFX与ctDNA间的相互作用,结果表明MXFX与DNA的结合是MXFX嵌入到DNA中相邻2个碱基对之间,属于嵌入结合模式。     

氧氟沙星和左氧氟沙星与DNA的相互作用研究

采用紫外光谱、荧光光谱、荧光偏振以及K₃Fe(CN)₆荧光猝灭实验研究了氧氟沙星(Ofloxacin,OFLX)和左氧氟沙星(Levofloxacin,L-OFLX)与小牛胸腺DNA(ctDNA)的相互作用差异性与作用模式。紫外光谱的结果表明,当向OFLX和L-OFLX溶液中加入ctDNA并且浓度增大时,OFLX和L-OFLX的吸收光谱都呈现略微的减色效应,但吸收峰位置没有发生偏移,L-OFLX的减色效应略强于OFLX的减色效应;从荧光光谱以及OFLX和L-OFLX的Scatchard方程,获得其键合常数分别为1.15×105 L·mol-1,3.75×105 L·mol-1,表明L-OFLX与ctDNA的相互作用要略强于OFLX与ctDNA的相互作用;荧光偏振实验、单双链ctDNA与药物作用实验、K₃Fe(CN)₆荧光猝灭实验都表明OFLX、L-OFLX与ctDNA的作用模式可能是沟槽结合。     

Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study

The interaction between the flavonoid hesperidin and bovine serum albumin (BSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. The results revealed that hesperidin caused the fluorescence quenching of BSA through a static quenching procedure. The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n, and apparent binding constant K_A, corresponding thermodynamic parameters ΔG^o, ΔH^o, ΔS^o at different temperatures were calculated. The distance r between donor (BSA) and acceptor (hesperidin) was obtained according to fluorescence resonance energy transfer. The effect of Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ on the binding constants between hesperidin and BSA were studied. The effect of hesperidin on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy and UV/Vis absorption spectroscopy.     

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K_SV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ΔH=-11.66 kJ·mol-1,ΔS=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。     

Spectroscopic and Molecular Modeling Studies of the Interaction Between 4′-O-(α-L-Oleandrosyl)daunorubicin and Human Serum Albumin and Its Analytical Application

4′-O-(α-L-Oleandrosyl)daunorubicin (ODNR) is a disaccharide analogue of daunorubicin with potent antitumor activity against leukemia cell line K562 cells and colon cancer cell line SW620 cells. In this paper, the binding interaction of ODNR with human serum albumin (HSA) was investigated under simulative physiological conditions by fluorescence spectroscopy in combination with UV absorption spectroscopy and molecular modeling method. A strong fluorescence quenching reaction of ODNR to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer equation. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to relevant fluorescent data and Van’t Hoff equation. The hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the results of molecular modeling study. In addition, the effects of other ions on the binding constants were also studied. Moreover, the synchronous fluorescence technique was successfully employed to determine the total proteins in serum, urine and saliva samples at room temperature under the optimum conditions with a wide linear range and satisfactory results.