Interaction of diethyl aniline methylphosphonate with DNA: Spectroscopic and isothermal titration calorimetry

In this study the diethyl aniline methylphosphonate (DAM) was synthesized, the interaction of DAM with ct-DNA has been investigated by fluorescence spectra, UV spectra, molecular modeling and isothermal titration calorimetry (ITC). The binding constant of DAM to ct-DNA calculated from both isothermal titration calorimetry and fluorescence spectra were found to be in the 10⁴ M⁻¹ range. According to the ethidium bromide displacement studies, UV spectra and isothermal titration calorimetry experimental results, it can be concluded that DAM is an intercalator that can slide into the G-C rich region of ct-DNA. Furthermore, the results obtained from molecular modeling corroborated the experimental results obtanied from spectroscopic and ITC investigations. At the same time, fluorescence spectra suggested that the mechanism of the interaction of DAM to ct-DNA was a static enhancing type. ITC data showed that ct-DNA/DAM binding is enthalpy controlled.     

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.     

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.

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.     

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.     

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。     

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.     

Methylene blue as a DNA probe for a comparative study of Cd, Pb and Cr ions binding to calf thymus DNA

Methylene blue (MB) was developed as a sensitive DNA probe for a comparative study of Cd²⁺, Pb²⁺ and Cr³⁺ ions binding with calf thymus DNA (ctDNA). The fluorescence intensity of the MB-ctDNA system increased dramatically when heavy metal ions (Cd²⁺, Pb²⁺ and Cr³⁺ ions) were added, which indicated that some of the bound MB molecules were released from the ctDNA base pairs. To compare the binding affinity of these three different heavy metal ions with ctDNA, the relationships between the fluorescence intensity of the MB-ctDNA-M (Metal ions) system and the concentration ratio of [M]/[DNA(p)] were investigated. The results showed that the order of the binding affinity of heavy metal ions with ctDNA had the following sequence: Cr³⁺> Cd²⁺>Pb²⁺. This order was further proved by the effects of heavy metal ions on the number of MB bound to ctDNA, the measurements of binding constants of these heavy metal ions to ctDNA, and the effects of heavy metal ions on the absorption of the MB-ctDNA system. In addition, the interaction mechanisms of Cd²⁺, Pb²⁺ and Cr³⁺ ions with ctDNA were also discussed in detail. These results indicated that their interaction mechanisms are related to the concentration ratios of heavy metal ions to DNA.     

Analysis of the binding mechanism between o-phenylenediamine and bovine hemoglobin by molecular spectroscopy and molecular modeling methods

Abstract

This study was aimed to reveal the binding mechanism between bovine hemoglobin and o-phenylenediamine by using molecular spectroscopy techniques and molecular modeling methods. The experimental results revealed that the fluorescence quenching mechanism was a combined dynamic and static quenching. The binding constant was (1.17?±?0.02)×10⁴ L/mol, and only a single binding site exists. The binding distance was 2.46?nm. The binding process was a spontaneous reaction, dominated by hydrophobic forces. The molecular docking simulations have also confirmed the results of the spectroscopic methods. The results reported here may significantly help understanding the interaction mechanism of o-phenylenediamine and hemoglobin.     

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.     

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％。     

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.     

Study of interaction of butyl p-hydroxybenzoate with human serum albumin by molecular modeling and multi-spectroscopic method

Study of the interaction between butyl p-hydroxybenzoate (butoben) and human serum albumin (HSA) has been performed by molecular modeling and multi-spectroscopic method. The interaction mechanism was predicted through molecular modeling first, then the binding parameters were confirmed using a series of spectroscopic methods, including fluorescence spectroscopy, UV-visible absorbance spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The thermodynamic parameters of the reaction, standard enthalpy ΔH⁰ and entropy ΔS⁰, have been calculated to be −29.52 kJ mol⁻¹ and −24.23 J mol⁻¹ K⁻¹, respectively, according to the Van’t Hoff equation, which suggests the van der Waals force and hydrogen bonds are the predominant intermolecular forces in stabilizing the butoben-HSA complex. Results obtained by spectroscopic methods are consistent with that of the molecular modeling study. In addition, alteration of secondary structure of HSA in the presence of butoben was evaluated using the data obtained from UV-visible absorbance, CD and FT-IR spectroscopies.     

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.     

Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach

The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4?~?11 Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10⁴–10⁵ M^?1) and negative free energy of interaction within the range of ?26.0?~??36.8 kJ mol^?1. The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.     

Characterization of the interaction between human lactoferrin and lomefloxacin at physiological condition: Multi-spectroscopic and modeling description

The interaction between lomefloxacin (LMF) and human lactoferrin (Hlf) was studied by using fluorescence, circular dichroism (CD) spectroscopic and molecular modeling measurements. By the fluorescence quenching results, it was found that the binding constant K_A=8.69×10⁵ L mol⁻¹, and number of binding sites n=1.75 at physiological condition. Experimental results observed showed that the binding of LMF to Hlf induced conformational changes of Hlf. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-Hlf complex by synchronous fluorescence. The quantitative analysis data of far-UV CD spectra from that of the α-helix 37.4% in free Hlf to 30.2% in the LMF-Hlf complex further confirmed that secondary structure of the protein was changed by LMF. Near-UV CD showed perturbations around tryptophan and tyrosine residues which involves perturbations of tertiary structure. The thermodynamic parameters like, ΔH° and ΔS°, have been calculated to be 63.411 kJ mol⁻¹ and 231.104 J mol⁻¹ K⁻¹, respectively. Thermodynamic analysis showed that hydrophobic interactions were the main force in the binding site but the hydrogen bonding and electrostatic interaction could not be excluded which in agreement with the result of molecular docking study. The distance r between donor and acceptor was obtained according to fluorescence resonance energy transfer (FRET) and found to be 1.78 nm. The interaction between LMF and Hlf has been verified as consistent with the static quenching procedure and the quenching mechanism is related to the energy transfer. Furthermore, the study of molecular modeling that LMF could bind to the α-helixes between Pro145-Asn152 and Phe167-Gln172 regions and hydrophobic interaction was the major acting force for the binding site, which was in agreement with the thermodynamic analysis.     

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.     

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.     

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.