Study on the interaction of amino phosphine ester derivatives with DNA by spectroscopy, modeling and calorimetry

The binding properties of amino phosphate ester derivatives, compound 1 and 2 with calf thymus DNA (CT-DNA) were investigated by UV spectra, fluorescence spectra, molecular modeling and isothermal titration calorimetry (ITC). The intrinsic binding constants K_b of compound 1 and 2 with CT-DNA were determined by fluorescence spectroscopy and ITC, respectively. The results indicated that the two compounds bind to CT-DNA with different binding affinity, which is in the order of compound 1 > compound 2. At the same time, fluorescence spectra suggested that the mechanism of the binding of the two compounds to CT-DNA is a static enhancing type. According to the ethidium bromide displacement experiments, UV spectra, molecular modeling and ITC studies, it can be concluded that compound 1 and 2 are intercalators that can slide into the G–C rich region of CT-DNA. Furthermore, ITC data showed that compound/DNA binding is enthalpy controlled.     

Investigation of the interaction between peanut agglutinin and synthetic glycopolymeric multivalent ligands

The interaction between synthetic glycoplymers bearing beta-D-galactose side groups and the lectin peanut agglutinin (PNA) was investigated by UV-difference spectroscopy and isothermal titration calorimetry (ITC). UV-difference spectroscopy indicated that the polymer-lectin interaction was stronger than that between PNA and either the corresponding monomer, D-galactose or D-lactose. The thermodynamics of binding (K, DeltaG, DeltaH, DeltaS and n) were determined from ITC data by fitting with a two-site, non-cooperative binding model. It was found that the glycopolymer displayed around a 50 times greater affinity for the lectin than the parent carbohydrate, and around 10 times greater than the monomer, on a valency-corrected basis. Binding was found to be entropically driven, and was accompanied by aggregation and precipitation of protein molecules. Furthermore, interesting differences between polymers prepared either from deacetylated monomers, or by deacetylation of pre-formed polymers, were found.     

Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.     

Generation-dependent host-guest interactions: solution States of polyglycerol dendrimers of generations 3 and 4 modulate the localization of a guest molecule

Host-guest interactions between polyglycerol dendrimers of generations?3 and 4 (PGD-G3 and G4) and 4-amino-3-hydroxynapthalene-2-sulphonic acid (AHSA) were investigated by fluorescence spectroscopy, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS). PGD-G3 molecules were found to form an associated state with an average diameter of 82.7?nm in aqueous solution, in which PGD-G3 provided a much more polar microenvironment than glycerol. PGD-G3 and AHSA interacted attractively, showing a binding constant of 5.3×10(5) M(-1) with a 2:1 stoichiometry. On the other hand, AHSA interacted with the periphery of PGD-G4, the majority of which existed as a unimer, forming a less polar microenvironment. The driving force of the interactions for PGD-G3 and -G4 were mainly enthalpically and entropically driven, respectively. The generation-dependent host-guest interactions were described in conjunction with thermodynamic parameters.     

咪唑型离子液体与牛血清蛋白的缔合特性

通过紫外-可见光谱、荧光光谱、同步荧光光谱、圆二色谱、衰减全反射红外光谱、负染-透射电镜、等温滴定微量热等实验方法系统地探讨了咪唑型离子液体与牛血清蛋白(BSA)的缔合特性.结果发现,离子液体[Bmim]Cl的加入使得BSA的紫外吸收强度增加,同时也会导致其荧光猝灭,并且这种猝灭是静态猝灭.同步荧光的研究结果表明,[Bmim]Cl分子可与蛋白质中接近色氨酸残基的区域发生相互作用,使蛋白质的构象和内部的疏水结构发生改变;负染色法透射电镜直观地显示了加入离子液体后形成的蛋白质-离子液体复合物结构逐渐变大;圆二色谱和衰减全反射红外光谱表明:在离子液体与BSA缔合过程中,离子液体的加入使得BSA二级结构中的α-螺旋和β-折叠的含量降低,从而引起蛋白质二级结构的变化;表面张力法和等温滴定微量热法进一步证实上述缔合作用为静电作用和疏水作用共同作用的结果,但离子液体的烷基链与BSA疏水内腔之间的疏水作用是离子液体与BSA缔合的主要驱动力.     

Generation‐Dependent Host–Guest Interactions: Solution States of Polyglycerol Dendrimers of Generations 3 and 4 Modulate the Localization of a Guest Molecule

Host–guest interactions between polyglycerol dendrimers of generations 3 and 4 (PGD‐G3 and G4) and 4‐amino‐3‐hydroxynapthalene‐2‐sulphonic acid (AHSA) were investigated by fluorescence spectroscopy, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS). PGD‐G3 molecules were found to form an associated state with an average diameter of 82.7 nm in aqueous solution, in which PGD‐G3 provided a much more polar microenvironment than glycerol. PGD‐G3 and AHSA interacted attractively, showing a binding constant of 5.3×10⁵ M ^?1 with a 2:1 stoichiometry. On the other hand, AHSA interacted with the periphery of PGD‐G4, the majority of which existed as a unimer, forming a less polar microenvironment. The driving force of the interactions for PGD‐G3 and ‐G4 were mainly enthalpically and entropically driven, respectively. The generation‐dependent host–guest interactions were described in conjunction with thermodynamic parameters.     

Study on interaction between poly(amidoamine) dendrimer and CdSe nanocrystal in chloroform

The binding of different categories of molecules to quantum dot has been studied for many years through different spectroscopic techniques to elucidate details of binding mechanism. In this work we present the results of the study of the interactions between CdSe and poly(amidoamine) dendrimer monitored by photoluminescence spectroscopy of CdSe in chloroform. Dendrimers with different terminal groups and different generations were used to bind with CdSe nanocrystal of different size. Significant differences in the values of binding constant Kb(n) and K(SV) were found in these experiments. The binding constant for poly(amidoamine) dendrimer of generation 4.0 is higher as compared to generation 3.5. The interaction of CdSe with poly(amidoamine) dendrimer shows an increase of binding constants with increasing dendrimer generation from 2.0 to 4.0, as well as with decreasing CdSe diameter. From HRTEM and FTIR analysis, we suggest that dendrimer/CdSe interactions are primarily hydrogen-bonding.     

季铵盐型阳离子表面活性剂与牛血清白蛋白的相互作用

本文合成并表征了三种不同烷基链长度的季铵盐型阳离子表面活性剂：N-十二烷基-N-（2-羟乙基）-N,N-二甲基溴化铵（DHDAB）、N-十四烷基-N-（2-羟乙基）-N,N-二甲基溴化铵（THDAB）、N-十六烷基-N-（2-羟乙基）-N,N-二甲基溴化铵（CHDAB）。采用荧光光谱法、紫外-可见光谱法、动态光散射法和等温滴定量热法对三种表面活性剂与牛血清白蛋白（BSA）的相互作用进行研究。荧光光谱研究表明,三种表面活性剂主要与BSA分子内的色氨酸残基发生相互作用,导致蛋白质的构象发生变化,且表面活性剂烷基链越长,与BSA的相互作用就越强。BSA荧光猝灭的主要原因是静态猝灭,紫外光谱实验同样验证了静态猝灭的存在。等温滴定量热法结果表明低浓度的表面活性剂与BSA主要发生静电作用和疏水作用而放热。动态光散射结果表明高浓度的表面活性剂会使BSA结构被破坏。本文揭示了表面活性剂与BSA相互作用的机理,为表面活性剂的广泛应用提供了理论基础。     

Study on the binding of puerarin to bovine serum albumin by isothermal titration calorimetry and spectroscopic approaches

The interaction of a flavonoid molecule (puerarin) with bovine serum albumin (BSA) was characterized by isothermal titration calorimetry (ITC), optical spectroscopic technique, and molecular modeling method under physiological conditions. The binding parameters for the reaction were calculated according to ITC experiments at different temperatures. The thermodynamic parameters, negative enthalpy changes (ΔH), and positive entropy (ΔS) indicated that the binding processes were entropically driven. The alterations of protein secondary structure in the presence of puerarin in aqueous solution were estimated by the evidences from FT-IR and CD spectroscopy with reductions of α-helices. On the basis of fluorescence resonance energy transfer (FRET) between excited tryptophan in BSA and BSA bound puerarin, the critical transfer distance and mean distance between tryptophan in BSA and puerarin were estimated.     

2,2,2-trifluoroethanol-induced molten globule state of concanavalin a and energetics of 8-anilinonaphthalene sulfonate binding: calorimetric and spectroscopic investigation

The interaction of 2,2,2-trifluoroethanol (TFE) with concanavalin A has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy at pH 2.5 and 5.2. All of the calorimetric transitions at both the pH values were found to be irreversible. In the presence of 4 mol kg(-1) TFE at pH 2.5, concanavalin A is observed to be in a partially folded state with significant loss of native tertiary structure. The loss of specific side chain interactions in the transition from native to the TFE-induced partially folded state is demonstrated by the loss of cooperative thermal transition and reduction of the CD bands in the aromatic region. Acrylamide quenching, 8-anilinonaphthalene sulfonate (ANS) binding, and energy transfer also suggest that in the presence of 4 mol kg(-1) TFE at pH 2.5 concanavalin A is in a molten globule state. ITC has been used for the first time to characterize the energetics of ANS binding to the molten globule state. ITC results indicate that the binding of ANS to the molten globule state and acid-induced state at pH 2.5 displays heterogeneity with two classes of non-interacting binding sites. The results provide insights into the role of hydrophobic and electrostatic interactions in the binding of ANS to concanavalin A. The results also demonstrate that ITC can be used to characterize the partially folded states of the protein both qualitatively and quantitatively.     

Eu(3+)-mediated polymerization of benzenetetracarboxylic acid studied by spectroscopy, temperature-dependent calorimetry, and density functional theory

Thermodynamic parameters for the complexation of Eu(3+) with pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid, BTC) as a model system for polymerizable metal-complexing humic acids were determined using temperature-dependent time-resolved laser-induced fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC). At low metal and ligand concentrations (<50 μM Eu(3+), <1 mM BTC), a 1:1 monomeric Eu-BTC complex was identified in the range of 25-60 °C. At elevated concentrations (>500 μM Eu(3+) and BTC) a temperature-dependent polymerization was observed, where BTC monomers are linked via coordinating shared Eu(3+) ions. The two methods lead to comparable thermodynamic data (ΔH = 18.5 ± 1.5/16.5 ± 0.1 kJ mol(-1); ΔS = 152 ± 5/130 ± 5 J mol(-1) K(-1); TRLFS/ITC) in the absence of polymerization. With the onset of polymerization, TRLFS reveals the water coordination number of the lanthanide, whereas calorimetry is superior in determining the thermodynamic data in this regime. Evaluating the heat uptake kinetics, the monomer and polymer formation steps could be separated by "time-resolved" ITC, revealing almost identical binding enthalpies for the sequential reactions. Structural features of the complexes were studied by Fourier-transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations showing predominantly chelating coordination with two carboxylate groups in the monomeric complex and monodentate binding of a single carboxylate group in the polymeric complex of the polycarboxylate with Eu(3+). The data show that pyromellitic acid is a suitable model for the study of metal-mediated polymerization as a crucial factor in determining the effect of humic acids on the mobility of heavy metals in the environment.     

Spectroscopic and Calorimetric Study of 2,2′-Dibipyridin Cu(II) Chloride Binding to Bovine β-Lactoglobulin

The interaction between β-lactoglobulin (BLG) and a newly synthesized Cu(II) complex (2,2′-dibipyridin Cu(II) chloride) was investigated by fluorescence spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC) at temperatures of 27 and 37 °C. The measured heat values of the BLG–Cu(II) complex interaction are reported and analyzed in terms of our previous extended solvation theory for calculating the binding and thermodynamic parameters for the interaction. The Cu(II) complex has a strong ability to quench the intrinsic fluorescence of BLG, to change the microenvironment of tryptophan residues, and to alter the tertiary structure of the protein. Far UV–CD results showed that the complex does not induce any significant changes in the secondary structure of BLG. However, binding of the Cu(II) complex to BLG leads to a significant change in the tertiary structure of BLG, increasing its hydrophobicity and inducing a partial unfolding. This agrees well with ITC data suggesting destabilization of the protein. This finding opens up a way to predict protein destabilization caused by ligand binding, using the extended solvation theory previously proposed.     

Affinity capillary electrophoresis and isothermal titration calorimetry for the determination of fatty acid binding with beta-cyclodextrin

Affinity capillary electrophoresis (ACE) and isothermal titration calorimetry (ITC) were used to investigate the binding interaction between several fatty acids (FAs) and beta-cyclodextrin (beta-CD). Within each method, steps taken to obtain accurate binding constants are discussed. The stoichiometry of interaction was revealed to be 1:1 regardless of FA chain length. The binding constants obtained using ACE were: octanoate, 6.4x10(2); 2-octenoate, 4.7x10(2); decanoate, 3.7x10(3); 9-decenoate, 1.8x10(3) and dodecanote, 1.4x10(4). The binding constants obtained from ITC were of the same order of magnitude, but were consistently greater than those from ACE. Thermodynamic data obtained using ITC are used to explain the observed trends in binding strength.     

Osteopontin: A Uranium Phosphorylated Binding‐Site Characterization

Herein, we describe the structural investigation of one possible uranyl binding site inside a nonstructured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phosphopeptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth‐inhibition site. Although thermodynamical aspects were investigated by using time‐resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X‐ray absorption fine structure (EXAFS) at the U L_III‐edge combined with attenuated total reflection Fourier transform infrared (ATR‐FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO₂²⁺/peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5–5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein.     

Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined through dynamic light scattering (DLS). Aggregation of drug/protein complexes was found as a result of a possible expansion of protein structure induced at high drug concentrations. In addition, the presence of free drug aggregates at concentrations below the drug critical micelle concentration was also detected.     

钌(II)配合物与酵母tRNA相互作用的等温滴定量热研究

The interaction of metal complex with RNA has been studied by isothermal titration calorimetry (ITC) for the first time. ITC experiments show that complex [Ru(phen)2MPIP]^2 {phen= 1,10-phenanthroline, MP[P-2-(4-methylphenyl)imidazo[4,5-f]-1, 10-phenanthroline} interacts with yeast tRNA in terms of a model for a singleset of identical sites through intercalation, which is consistent with our previous observation obtained from spectroscopic methods, and this binding process was driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, suggesting that ITC is an effective method for deep studying the interactions of metal complexes with RNA.     

Energetics of binding and protein unfolding upon amphiphilic drug complexation with a globular protein in different aqueous media

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by zeta-potential, isothermal titration calorimetry (ITC), UV-vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. zeta-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from zeta-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV-vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water (DeltaG(w)(degrees)) and in a hydrophobic environment (DeltaG(hc)(degrees)). Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.     

Implication of Threonine-Based Ionic Liquids on the Structural Stability,Binding and Activity of Cytochrome c

Ionic liquids (ILs) are useful in pharmaceutical industries and biotechnology as alternative solvents or sources for protein extraction and purification, preservation of biomolecules and for regulating the catalytic activity of enzymes. However, the binding mechanism, the non-covalent forces responsible for protein-IL interactions and dynamics of proteins in IL need to be investigated in depth for the effective use of ILs as alternatives. Herein, we disclose the molecular level understanding of the structural intactness and reactivity of a model protein cytochrome c (Cyt c) in biocompatible threonine-based ILs with the help of experimental techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy, transmission electron microscopy (TEM) as well as molecular docking. Hydrophobic and electrostatic forces are responsible for the structural and conformational integrity of Cyt c in IL. The ITC experiments revealed the Cyt c-IL binding free energies are in the range of 10–14 kJ/mol and the molecular docking studies demonstrated that ILs interact at the surfaces of Cyt c. The results look promising as the ILs used here are non-toxic and biocompatible, and thus may find potential applications in structural biology and biotechnology.     

Synthesis of carbosilane dendrimers having peripheral mannose and mannobiose

The mannose monosaccharide derivative, acetylthiopropyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (Man), and the mannobiose derivative, acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside (α-1,3-Man), were synthesized respectively. These mannose derivatives were introduced into carbosilane dendrimer scaffolds of the zero and first generations. As a result, six carbosilane dendrimers were functionalized by Man and α-1,3-Man. Isothermal titration microcalorimetry was done to determine binding assay between mannose moieties of carbosilane dendrimer and concanavalin A. It was found that carbosilane dendrimers bound more efficiently to concanavalin A than free mannose (Me-α-Man) and mannobiose (Me-α-1,3-Man).     

环糊精对阿斯巴甜的甜感增强作用及二者相互作用热力学

目前环糊精（CD）对阿斯巴甜（ASP）甜感强度的影响研究主要集中在环糊精对阿斯巴甜的稳定性研究。我们认为CD对ASP甜感强度的提升与其和ASP的结合常数有一定的关系。本文选择了五种环糊精,α-环糊精（α-CD）、β-环糊精（β-CD）、γ-环糊精（γ-CD）、羟丙基-β-环糊精（HP-β-CD）、甲基-β-环糊精（Met-β-CD）,研究了这些环糊精存在下ASP的感官甜度的变化及二者的相互作用。结果表明,β-CD可以明显提升ASP的甜感强度。等温滴定量热（ITC）和荧光光谱对ASP与CDs结合过程亲和力的研究表明,ASP与β-CD的结合是自发的,并且具有最大的结合常数。差示扫描量热（DSC）、核磁共振（¹H NMR）以及傅里叶变换红外（FT-IR）揭示了其结合过程的机制。本研究对理解甜味剂甜感强度与热力学结合常数的关系具有重要的意义,也为基于结合常数筛选风味保持剂的方法提供有益的基础。