Study of the Enzymatic Activity of Arthrobacter ureafaciens Neuraminidase by Isothermal Titration Calorimetry

Isothermal titration calorimetry was used to determine the enzymatic activity and thermodynamic activation parameters of Arthrobacter ureafaciens sialidase with the sialyl substrates α‐2,3‐, α‐2,6‐sialyllactoses and α‐2,8‐sialic acid dimer. By monitoring the heat released during hydrolysis, the Michaelis constant (K_m), catalytic rate constant (k_cat), activation energy, activation Gibbs energy, enthalpy, and entropy for different monovalent sialyl conjugates were calculated and found to be consistent with those derived by chromatographic or colorimetric assays. The observed decreases in the activation energy and transition entropy of sialyllactoses were larger than the Michaelis activation parameters of lactose‐free di‐sialic acid because of the specific enzyme activity of A. ureafaciens sialidase.     

Multivalent neuraminidase hydrolysis resistant triazole-sialoside protein conjugates as influenza-adsorbents

Triazole-sialoside tailored proteins with high hemagglutinin (HA) and neuraminidase (NA) binding affinity are prepared. Dynamic light scattering shows that these pseudo-sialylated proteins are ideal virus capture macromolecules.     

Controlled complexation of plasmid DNA with cationic polymers: effect of surfactant on the complexation and stability of the complexes

The aggregation of the cationic polymer-plasmid DNA complexes of two commonly used polymers, polyethyleneimine (PEI) and poly-l-lysine (PLL) were systematically compared. The complexation was studied in 5% glucose solution at 25 degrees C using dynamic light scattering and isothermal titration calorimetry. The aggregation of the complexes was controlled by addition of the surfactant polyoxyethylene stearate (POES). The stability of the complexes was evaluated using dextran sulphate (DS) as relaxing agent. The relaxation of the complexes in the presence of DS was studied using agarose gel electrophoresis. This study elucidates the role of surfactant in controlling the size of the PEI/pDNA complex and reveals the differences of the two polymers as complexing agents.     

A predictive, simple and rapid method for thermodynamic study on the binding of copper ion with Alzheimer''s amyloid β peptide

The interaction of CuC12 to the first 16 residues of the Alzbeimer's amyliod β peptide, Aβ(1-16) was studied by isothermal titration calorimetry at pH 7.2 and 37 ℃ in aqueous solution.     

Rapid and efficient screening of adsorbent for oligopeptide using molecular docking and isothermal titration calorimetry

A rapid and efficient method based on molecular docking and isothermal titration calorimetry (ITC) was developed to identify effective adsorbents for the target peptide Ser‐Glu‐Ala‐Asp‐His (SEADH). Preliminary screening of five candidate adsorbents using molecular docking revealed that three suitable structures (A1, A2, and A3) either with or without coordination of Zn²⁺ should be effective. The three selected structures were then prepared and further screened by evaluating their affinities for the peptide SEADH using ITC. The screening results revealed that the adsorbent A2 coordinated with Zn²⁺ was the best adsorbent, and subsequent static adsorption experiments confirmed the reliability of the screening method. Further ITC analysis, combined with molecular docking, was performed to provide the possible adsorption mechanism.     

Synthesis, solid-state structures and metal ion complexation behavior of 2-picolyloxy lariat ethers

Three new 2-picolyloxy lariat ether compounds are prepared and solid-state structures are determined for two of them. Complexation behavior for a series of lariat ethers with pyridyl-containing side arms toward alkali metal cations and Ag⁺ is assessed by two-phase metal picrate extraction from aqueous solutions into chloroform and in homogeneous solutions by isothermal titration calorimetry in methanol.     

合成受体与模型磷酸化肽的相互作用机理

为了研究基于Zn2+-二甲基吡啶胺及胍羰基吡咯基团的配位型受体Zn Dpa G与磷酸化肽的相互作用机制,选取具有不同序列的磷酸化肽作用模型,采用等温滴定微量热法考察了Zn Dpa G与磷酸化肽的结合常数,研究了模型肽中磷酸基团的数量、密度及位置等因素对多肽与受体间结合强度的影响.结果表明,Zn Dpa G受体对双磷酸化肽结合能力显著高于单磷酸化肽,其结合常数可提高10~40倍,2个磷酸基团的距离越近,结合作用越强;而磷酸基团的位置显著影响受体与单磷酸化肽的结合强度.本研究结果为进一步优化磷酸化肽受体结构设计,实现肽与受体间高选择性识别提供了一定的理论依据.     

QSAR analyses on avian influenza virus neuraminidase inhibitors using CoMFA, CoMSIA, and HQSAR

The recent wide spreading of the H5N1 avian influenza virus (AIV) in Asia, Europe and Africa and its ability to cause fatal infections in human has raised serious concerns about a pending global flu pandemic. Neuraminidase (NA) inhibitors are currently the only option for treatment or prophylaxis in humans infected with this strain. However, drugs currently on the market often meet with rapidly emerging resistant mutants and only have limited application as inadequate supply of synthetic material. To dig out helpful information for designing potent inhibitors with novel structures against the NA, we used automated docking, CoMFA, CoMSIA, and HQSAR methods to investigate the quantitative structure-activity relationship for 126 NA inhibitors (NIs) with great structural diversities and wide range of bioactivities against influenza A virus. Based on the binding conformations discovered via molecular docking into the crystal structure of NA, CoMFA and CoMSIA models were successfully built with the cross-validated q (2) of 0.813 and 0.771, respectively. HQSAR was also carried out as a complementary study in that HQSAR technique does not require 3D information of these compounds and could provide a detailed molecular fragment contribution to the inhibitory activity. These models also show clearly how steric, electrostatic, hydrophobicity, and individual fragments affect the potency of NA inhibitors. In addition, CoMFA and CoMSIA field distributions are found to be in well agreement with the structural characteristics of the corresponding binding sites. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing novel potent NA inhibitors.     

The synthesis and characterization of lipophilic peptide-based carriers for gene delivery

Lipophilic carriers have shown great potential in improving the delivery of gene therapeutics. We have synthesized positively charged peptide-based carriers including lipoamino acids. The carriers were shown to interact with DNA by performing isothermal titration calorimetry and particle size and zeta potential experiments. An exothermic reaction resulted from the titration of carrier into DNA. The particle sizes of the carrier/DNA complexes varied over the different charge ratios from 200-800 nm. The zeta potential was negative at a low charge ratio but positive when the amount of carrier was increased. The utilisation of lipophilic carriers is a promising approach to improve the bioavailability of gene delivery.     

古细菌RNase HII与金属离子结合的热力学研究

RNase H是一种专一性水解RNA:DNA杂合链中RNA链的核糖核酸酶,它广泛存在于从原核生物到人的生物体中.本文通过等温滴定量热技术研究了Mg2＋,Mn2＋和Ca2＋与一种古细菌Methanococcus jannaschii中的II型RNase H结合的热力学.首次用这种方法获得了这一结合过程的热力学参数.并证实了这些金属离子与RNase HII按1∶1结合.为RNase HII酶反应机理和折叠研究提供了重要信息.     

Application of a new method for data analysis of isothermal titration calorimetry in the interaction between human serum albumin and Ni

The interaction of human serum albumin (HAS) with divalent nickel ion was studied by isothermal titration calorimetry (ITC) in 30 mM Tris buffer, pH 7.0. There is a set of eight identical and independent binding sites for nickel ions on the protein at the temperature of 300 K. A new calorimetric data analysis allows the determination of the complete set of thermodynamic parameters. The binding isotherm for nickel-HSA interaction is easily obtained by carrying out two different ITC experiments. In the first experiment, the enthalpy of binding for one mole of nickel ion to one mole of binding site on HSA (ΔH=−36.5 kJ) is obtained, and is used in a second experiment to determine the binding isotherm and to find the number of binding sites (g=8) and the equilibrium constant (K=0.57 μM⁻¹).     

A New Approach for Thermodynamic Studies on Binding of Some Metal Ions with Human Growth Hormone

A thermodynamic study on the interaction between Mg²⁺, Ni²⁺ and Co²⁺ ions (M²⁺) and human growth hormone (hGH) was made at 27 °C in aqueous NaCl solutions using isothermal titration calorimetry. Gholamreza Rezaei Behbehani’s solvation model (GRB) was used to model the enthalpies of M²⁺+hGH interactions over the studied range of metal ion concentrations. The solvation parameters derived from the solvation model were attributed to a structural change of hGH due to its interactions with the metal ion.     

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.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions

The application of isothermal titration calorimetry (ITC) was tested towards measurements of enzyme kinetics in complex solutions containing high concentrations of proteins. Such investigations are important, due to the increasing interest in biochemical reactions in physiological relevant media as well as the application of enzymes in industrial processes. In contrast to spectral methods, measurements performed with ITC, are independent of the optical properties of solutions, making it possible to measure enzyme kinetics in concentrated solutions of macromolecules. In this study the kinetic properties of hexokinase was investigated in concentrated protein solutions (BSA). It was found, that the quality of the measured kinetic data was independent of protein concentration in the investigated range (0-250 mg BSA ml⁻¹). All results could be accounted for by Michaelis-Menten's approach and both k_cat and K_M decreased with increasing protein concentrations. The decrease in K_M with increasing protein concentration was ascribed to an increase in the ratio of activity coefficients between the native enzyme and the enzyme-substrate complex. The decrease in k_cat with increasing protein concentrations indicates that crowding by BSA effect the conformational changes/rehydration that accompanies catalysis and/or diffusion of product from the enzyme-product complex. The methodology is discussed together with an analysis of the experimental results.     

基于动态光散射技术的含乳糖组装体与蛋白质相互作用

采用非共价键合胶束(NCCM)的组装策略, 制备了表面为糖聚合物的纳米粒子, 利用动态光散射技术, 实时跟踪了在溶液中糖与蛋白相结合的动力学数据, 并以聚集体形成的速度来体现不同的蛋白对相同组装体的结合能力强弱. 实验结果表明, 对表面覆盖100%乳糖的组装体, 各蛋白的结合能力表现为SBA?LecA≈Galectin 3>ECA≈Galectin 1, 表明蛋白的糖结合位点个数及其空间分布对多价作用的动力学有较大影响. 基于此模型, 对人半乳凝素-3与糖结合的方式进行了初步探究.     

Characterization of recognition sites for diethyl 4-nitrobenzylphosphonate, an organophosphate pesticide analogue

In a first step towards chemical sensors using molecular imprinted materials, the complexing characteristics of diethyl 4-nitrobenzylphosphonate, an organophosphate pesticide analogue, have been studied. Two molecules have been assessed as potential interacting moieties, specifically a fluoroalcohol and an aromatic acid. The interactions have been first characterized by regular methods, such as ¹H, ³¹P NMR and IR spectroscopy. These showed a stoichiometry 1/1 for both complexes and association constants, respectively, close to 40 ± 10 and 12 ± 2 M⁻¹. In a second step, isothermal titration calorimetry was used and a method was developed to obtain low-association constants. The association constant could be obtained for the fluoroalcohol ligand and was found equal to 63 ± 0.7 M⁻¹. For the acidic molecule, an appropriate model could not be found, preventing the evaluation of this constant.     

Thermodynamic study on the interaction between anti-tumor drug tegafur and human serum albumin

The changes of thermodynamic properties of the system on interaction between tegafur and human serum albumin (HSA) and the changes of secondary structure units of HSA in the system at 298.15 K have been investigated by the Nano-Watt-Scale isothermal titration calorimetry (ITC), the Langmuir’s binding model and the circular dichroism (CD) spectrometry.     

Temperature dependence of the interaction of cholate and deoxycholate with fluid model membranes and their solubilization into mixed micelles

The interaction of bile salts with model membranes composed of soybean phosphatidylcholine (SPC) and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using high sensitivity isothermal titration calorimetry (ITC). The partitioning and incorporation of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) from an aqueous phase (pure water or 0.1 M NaCl) into fluid bilayer vesicles was studied as a function of temperature and ionic strength. The thermodynamic parameters of partitioning were determined with a model taking electrostatic interactions into account. In addition, the solubilization of SPC and POPC vesicles with NaC and NaDC as a function of temperature was also studied by ITC and the phase diagrams for the vesicle to micelle transition at two different temperatures were established. Unsaturated phospholipids require higher amounts of detergent to be transformed into micelles compared to saturated phospholipids. In addition, the width of the coexistence region of mixed micelles and mixed vesicles is larger for phosphatidylcholines with unsaturated chains. A comparison of NaDC with NaC shows the higher solubilization effectiveness of NaDC in agreement with its lower cmc. Furthermore, increasing the ionic strength decreases the amount of bile salt necessary for the formation of mixed micelles, which is also expected from the decrease of the cmc with ionic strength due to the shielding of the charges of the bile salts.