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Isothermal titration calorimetry (ITC) directly measures heat evolved in a chemical reaction to determine equilibrium binding properties of biomolecular systems. Conventional ITC instruments are expensive, use complicated design a...
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Isothermal titration calorimetry (ITC) directly measures heat evolved in a chemical reaction to determine equilibrium binding properties of biomolecular systems. Conventional ITC instruments are expensive, use complicated design and construction, and require long analysis times. Microfabricated calorimetric devices are promising, although they have yet to allow accurate, quantitative ITC measurements of biochemical reactions. This paper presents a microfabrication-based approach to integrated, quantitative ITC characterization of biomolecular interactions. The approach integrates microfabricated differential calorimetric sensors with microfluidic titration. Biomolecules and reagents are introduced at each of a series of molar ratios, mixed, and allowed to react. The reaction thermal power is differentially measured, and used to determine the thermodynamic profile of the biomolecular interactions. Implemented in a microdevice featuring thermally isolated, well-defined reaction volumes with minimized fluid evaporation as well as highly sensitive thermoelectric sensing, the approach enables accurate and quantitative ITC measurements of protein-ligand interactions under different isothermal conditions. Using the approach, we demonstrate ITC characterization of the binding of 18-Crown-6 with barium chloride, and the binding of ribonuclease A with cytidine 2'-monophosphate within reaction volumes of approximately 0.7 mu L and at concentrations down to 2 mM. For each binding system, the ITC measurements were completed with considerably reduced analysis times and material consumption, and yielded a complete thermodynamic profile of the molecular interaction in agreement with published data. This demonstrates the potential usefulness of our approach for biomolecular characterization in biomedical applications. (C) 2015 Elsevier B.V. All rights reserved.
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Inclusion complex formation between β-cyclodextrin (β-CD) and suitable guest molecules has frequently been exploited to design self-assembled polymer networks. In this paper, we report on hydrogels composed of eight-armed poly(e...
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Inclusion complex formation between β-cyclodextrin (β-CD) and suitable guest molecules has frequently been exploited to design self-assembled polymer networks. In this paper, we report on hydrogels composed of eight-armed poly(ethylene glycol) (PEG) grafted with β-CD (8armPEG20k-CD), adamantane (8armPEG20k-ad) or cholesterol (8armPEG20k-chol). Mixtures of 8armPEG20k-CD and 8armPEG20k-ad showed viscous behavior (G″ > G′); 8armPEG20k-CD and 8armPEG20k-chol formed elastic hydrogels (G′ > G″). To study the interaction of adamantane and cholesterol grafted PEG with β-CD, linear model compounds (mPEG5k-ad and mPEG5k-chol) were synthesized. Isothermal titration calorimetry (ITC) experiments showed a higher association constant for inclusion complexes formed with mPEG5k-chol (47,000 ± 1650) than for those formed with mPEG5k-ad (30,000 ± 900). Fluorescence spectroscopy and dynamic light scattering (DLS) measurements further showed the ability of mPEG5k-chol to self-assemble into micelles. Self-assembly of 8armPEG20k-chol further increased the strength of the formed hydrogels. Altogether, this study contributes to a better understanding of cyclodextrin based biomaterials.
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Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of d...
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Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of data analysis compare alternative methods for determining ligand binding enthalpy and analyze potential sources of error in the experimental measurement of other thermodynamic parameters. Several reports examine issues concerning drug design and the correlation of thermodynamic and X-ray structural data. New instruments allow volumetric effects in biochemical systems to be evaluated calorimetrically and to substantially expand the throughput of differential scanning calorimetry measurements in drug discovery and other high-throughput applications. [References: 26]
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Isothermal Titration Calorimetry (ITC) was used to study the thermodynamics of hybridization on DNA-functionalized colloidal gold nanoparticles. When compared to the thermodynamics of hybridization of DNA that is free in solution,...
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Isothermal Titration Calorimetry (ITC) was used to study the thermodynamics of hybridization on DNA-functionalized colloidal gold nanoparticles. When compared to the thermodynamics of hybridization of DNA that is free in solution, the differences in the values of the Gibbs free energy of reaction, Δ_rG°, the enthalpy, Δ_rH°, and entropy, Δ_rS°, were small. The change in Δ_rG° between the free and bound states was always positive but with statistical significance outside the 95% confidence interval, implying the free DNA is slightly more stable than when in the bound state. Additionally, ITC was also able to reveal information about the binding stoichiometry of the hybridization reactions on the DNA-functionalized gold nanoparticles, and indicates that there is a significant fraction of the DNA on gold nanoparticle surface that is unavailable for DNA hybridization. Furthermore, the fraction of available DNA is dependent on the spacer group on the DNA that is used to span the gold surface from that to the probe DNA.
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Dimerization of the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS) in aqueous media have been studied by isothermal titration calorimetry (ITC). ITC experiments carried out at different pHs show that dimerization c...
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Dimerization of the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS) in aqueous media have been studied by isothermal titration calorimetry (ITC). ITC experiments carried out at different pHs show that dimerization constants are highly pH dependent, decreasing their values with increasing pH. No dimerization is detected over pH 7. Analyzing the dependence of K_(dim) on pH, using a model that only considers dimers between zwitterionic molecules of ANS, a value of 5.6 for the pK of anilinium moiety is obtained. It is in agreement with the pK determined spectrophotometrically. The dimerization process is enthalpically disfavored and entropically driven at all pH and temperatures studied, indicating that hydrophobic effect has an important role on the formation of dimers. Although dimerization constants are low, dimerization equilibria must be taken into account when the energetics of the interaction of ANS to a protein is studied at pH below 7.
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Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A_2 (hsPLA_2GIIA) was invest...
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Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A_2 (hsPLA_2GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration cal-orimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA_2GIIA, and indicates that suramin represents a novel class of phospholipase A_2 inhibitor.
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Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A_2 (hsPLA_2GIIA) was invest...
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Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A_2 (hsPLA_2GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration cal-orimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA_2GIIA, and indicates that suramin represents a novel class of phospholipase A_2 inhibitor.
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Interaction of gold nanorods with cysteine as well as 3-mercaptopropionic acid (MPA) has been investigated by isothermal titration calorimetry (ITC), in combination with electronic absorption spectroscopy and electron microscopy. ...
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Interaction of gold nanorods with cysteine as well as 3-mercaptopropionic acid (MPA) has been investigated by isothermal titration calorimetry (ITC), in combination with electronic absorption spectroscopy and electron microscopy. The assembly process with MPA shows two steps, the first due to the binding of MPA to gold nanorods through the sulfur atom, and the second due to assembly of the MPA capped gold nanorods through the formation of cyclic hydrogen bonded dimers between the MPA molecules. In the case of cysteine only a single step is observed in ITC, due to the binding of the molecules to gold nanorods.
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Interaction of gold nanorods with cysteine as well as 3-mercaptopropionic acid (MPA) has been investigated by isothermal titration calorimetry (ITC), in combination with electronic absorption spectroscopy and electron microscopy. ...
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Interaction of gold nanorods with cysteine as well as 3-mercaptopropionic acid (MPA) has been investigated by isothermal titration calorimetry (ITC), in combination with electronic absorption spectroscopy and electron microscopy. The assembly process with MPA shows two steps, the first due to the binding of MPA to gold nanorods through the sulfur atom, and the second due to assembly of the MPA capped gold nanorods through the formation of cyclic hydrogen bonded dimers between the MPA molecules. In the case of cysteine only a single step is observed in ITC, due to the binding of the molecules to gold nanorods. (c) 2007 Elsevier B.V. All rights reserved.
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Hypothesis: Amphiphilic molecules spontaneously adsorb to fluid polar-nonpolar interfaces. The time scale of such adsorption depends on the molecular size and structure of the solute. This process should be accompanied by a power ...
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Hypothesis: Amphiphilic molecules spontaneously adsorb to fluid polar-nonpolar interfaces. The time scale of such adsorption depends on the molecular size and structure of the solute. This process should be accompanied by a power heat exchange that could be detected by commercial isothermal calorimeters.
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