Isothermal titration calorimetry for chiral chemistry

One of the most powerful techniques that are currently available to measure thermodynamic parameters such as enthalpy (ΔH), Gibbs free energy (ΔG), entropy changes (ΔS), and binding affinity in chemical reactions is isothermal titration calorimetry (ITC). Recent advances in instrumentation have facilitated the development of ITC as a very essential analytical tool in biology and chemistry. In this article, we will focus on a review of the literature on the application of ITC for the study of chiral systems and chiral interactions. We present studies in which the ITC technique is used to study chiral interactions, for instance in chiral solutions, chiral organometallic complexes, guest‐host chiral binding interactions, and biological macromolecules. Finally, we put strong emphasis on the most recent application of ITC for the study of chirality in nanosystems and at the nanoscale.     

Metastable and stable states of xanthan polyelectrolyte complexes studied by atomic force microscopy

The compaction of the semiflexible polysaccharide xanthan with selected multi- and polyvalent cations was studied. Polyelectrolyte complexes prepared at concentrations of 1-2 microg/ml were observed by tapping mode atomic force microscopy. High-molecular-weight xanthan compacted with chitosan yields a blend of mainly toroidal and metastable structures and a small fraction of rod-like species. Polyelectrolyte complexes of xanthan with polyethylenimine and trivalent chromium yielded similar structures or alternatively less well packed species. Racquet-type morphologies were identified as kinetically trapped states occurring on the folding path toward the energetically stable state of the toroids. Thermal annealing yielded a shift of the distribution of xanthan-chitosan morphologies toward this stable state. Ensembles of toroidal and rod-like morphologies of the xanthan-chitosan structures, collected using an asphericity index, were analyzed. The mean height of the toroids increased upon heating, with a selective increase in the height range above 2 nm. It is suggested that the observed metastable structures are formed from the high-molecular-weight fraction of xanthan and that these are driven toward the toroidal state, being a low-energy state, following annealing. Considered a model system for condensation of semiflexible polymers, the compaction of xanthan by chitosan captures the system at various stages in the folding toward a low-energy state and thus allows experimental analyses of these intermediates and their evolution.     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

A survey of the year 2003 literature on applications of isothermal titration calorimetry

Over the last decade isothermal titration calorimetry (ITC) has developed from a specialist method which was largely restricted in its use to dedicated experts, to a major, commercially available tool in the arsenal directed at understanding molecular interactions. The number of those proficient in this field has multiplied dramatically, as has the range of experiments to which this method has been applied. This has led to an overwhelming amount of new data and novel applications to be assessed. With the increasing number of publications in this field comes a need to highlight works of interest and impact. In this overview of the literature we have attempted to draw attention to papers and issues for which both the experienced calorimetrist and the interested dilettante hopefully will share our enthusiasm.     

A survey of the year 2002 literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is becoming widely accepted as a key instrument in any laboratory in which quantification of biomolecular interactions is a requisite. The method has matured with respect to general acceptance and application development over recent years. The number of publications on ITC has grown exponentially over the last 10 years, reflecting the general utility of the method. Here all the published works of the year 2002 in this area have been surveyed. We review the broad range of systems to which ITC is being directed and classify these into general areas highlighting key publications of interest. This provides an overview of what can be achieved using this method and what developments are likely to occur in the near future.     

Applications of isothermal titration calorimetry in protein science

During the past decade, isothermal titration calorimetry (ITC) has developed from a specialist method for understanding molecular interactions and other biological processes within cells to a more robust, widely used method. Nowadays, ITC is used to investigate all types of protein interactions, including protein-protein interactions, protein-DNA/RNA interactions, protein-small molecule interactions and enzyme kinetics; it provides a direct route to the complete thermodynamic characterization of protein interactions. This review concentrates on the new applications of ITC in protein folding and misfolding, its traditional application in protein interactions, and an overview of what can be achieved in the field of protein science using this method and what developments are likely to occur in the near future. Also, this review discusses some new developments of ITC method in protein science, such as the reverse titration of ITC and the displacement method of ITC.     

Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔH_app). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H₂O₂ production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.     

Comparative study of the bindings between 3-phenyl-1H-indazole and five proteins by isothermal titration calorimetry,spectroscopy and docking methods

Abstract

In this study, the interaction between 3-phenyl-1H-indazole (1a) and the fat mass and obesity-associated (FTO) protein was confirmed by isothermal titration calorimetry (ITC). The structure feature of 1a was different from our previously reported FTO inhibitors (radicicol, N-CDPCB and CHTB); the Cl and diol group in structure motif is critical for inhibitors to bind to FTO. In order to test whether there is specificity for the interaction between FTO and 1a, the interactions between 1a and four important proteins (human serum albumin (HSA), pepsin, catalase and trypsin) were investigated by ITC, spectroscopy and molecular docking methods. ITC results showed spontaneous exothermic reactions occurring between 1a and the proteins except trypsin under investigated conditions. The order of the binding affinity of 3-phenyl-1H-indazole is catalase?>?HSA?>?FTO?>?pepsin. Comparison between ITC and spectral results was made. This work will provide the basis for the design of novel inhibitors for FTO. Abbreviations CAT catalase

DMSO dimethyl sulfoxide

FTO fat mass and obesity-associated protein

HSA human serum albumin

Pep pepsin

Try trypsin

Communicated by Ramaswamy H. Sarma     

Survey of the year 2004: literature on applications of isothermal titration calorimetry

The market for commercially available isothermal titration calorimeters continues to grow as new applications and methodologies are developed. Concomitantly the number of users (and abusers) increases dramatically, resulting in a steady increase in the number of publications in which isothermal titration calorimetry (ITC) plays a role. In the present review, we will focus on areas where ITC is making a significant contribution and will highlight some interesting applications of the technique. This overview of papers published in 2004 also discusses current issues of interest in the development of ITC as a tool of choice in the determination of the thermodynamics of molecular recognition and interaction.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Isothermal titration calorimetry — A new method for the quantification of microbial degradation of trace pollutants

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2-3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4-(2,4-dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis-Menten kinetics. The half-saturation constant k_D and the degradation rate r_D^Max were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.     

Toxic effects of chrysoidine on human serum albumin: isothermal titration calorimetry and spectroscopic investigations

Chrysoidine is widely used in industry as a type of azo dye, and is sometimes used illegally as a food additive despite its potential toxicity. Human serum albumin (HSA) is one of the most important proteins in blood plasma and possesses major physiological functions. In the present study, the conformational and functional effects of chrysoidine on HSA were investigated by isothermal titration calorimetry (ITC), multiple spectroscopic methods, a molecular docking study and an esterase activity assay. Based on the ITC results, the binding stoichiometry of chrysoidine to HSA was estimated to be 1.5:1, and was a spontaneous process via a single hydrogen bond. The binding of chrysoidine to HSA induced dynamic quenching in fluorescence, and changes in secondary structure and in the microenvironment of the Trp‐214 residue. In addition, the hydrogen bond (1.80 Å) formed between the chrysoidine molecule and the Gln‐211 residue. The esterase activity of HSA decreased following the addition chrysoidine due to the change in protein structure. This study details the direct interaction between chrysoidine and HSA at the molecular level and the mechanism for toxicity as a result of the functional changes induced by HSA structural variation upon binding to chrysoidine in vitro. This study provides useful information towards detailing the transportation mechanism and toxicity of chrysoidine in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Explicit formulation of titration models for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) produces a differential heat signal with respect to the total titrant concentration. This feature gives ITC excellent sensitivity for studying the thermodynamics of complex biomolecular interactions in solution. Currently, numerical methods for data fitting are based primarily on indirect approaches rooted in the usual practice of formulating biochemical models in terms of integrated variables. Here, a direct approach is presented wherein ITC models are formulated and solved as numerical initial value problems for data fitting and simulation purposes. To do so, the ITC signal is cast explicitly as a first-order ordinary differential equation (ODE) with total titrant concentration as independent variable and the concentration of a bound or free ligand species as dependent variable. This approach was applied to four ligand-receptor binding and homotropic dissociation models. Qualitative analysis of the explicit ODEs offers insights into the behavior of the models that would be inaccessible to indirect methods of analysis. Numerical ODEs are also highly compatible with regression analysis. Since solutions to numerical initial value problems are straightforward to implement on common computing platforms in the biochemical laboratory, this method is expected to facilitate the development of ITC models tailored to any experimental system of interest.     

Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry

Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10(7) -10(8) M(-1) and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab')(2) fragment with a Ka value of 5.1 × 10(5) M(-1) ; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes.     

应用单针注射等温微量量热滴定法检测尿素水解菌对Hg2+的耐受性

【目的】利用单针注射等温微量量热滴定法(SIITC)检测尿素水解菌对Hg~(2+)的耐受性。【方法】与传统的细胞生长曲线法和基于尿素水解的p H变化曲线相对比,应用SIITC研究耐盐尿素水解菌Staphylococcus succinus对不同浓度Hg~(2+)的耐受性,通过相关性分析验证SIITC检测细菌对重金属胁迫耐受性的可行性。【结果】S.succinus菌吸附水解尿素的最大热量交换速率和累积热量值随Hg~(2+)浓度升高而降低。最大热量交换速率由不含Hg~(2+)的-45μJ/s降至10μmol/L Hg~(2+)处理的-20μJ/s,吸收热量由-1 291.90μJ下降到-535.75μJ;不同浓度Hg~(2+)胁迫下S.succinus菌吸附水解尿素的反应热量值与溶液p H以及OD_(600)的相关系数r分别达到0.928和0.955。【结论】SIITC可用来检测Hg~(2+)对尿素水解菌S.succinus的毒性。     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.     

Recent trends and some applications of isothermal titration calorimetry in biotechnology

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.