Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis–Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37 °C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis–Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.     

Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

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.     

Enthalpy of the B-to-Z conformational transition of a DNA oligonucleotide determined by isothermal titration calorimetry

The influence of high concentrations of Na(+) or [Co(NH(3))(6)](3+) on the conformation of two related DNA oligomers was investigated by circular dichroism spectropolarimetry (CD), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC). As revealed by CD, DNA oligomers, (dC-dG)(4) and (dm(5)C-dG)(4), both form right-handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na(+) at 25 degrees C. However, at 2.0 M Na(+) or 200 microM [Co(NH(3))(6)](3+), (dm(5)C-dG)(4) assumes a left-handed double helical structure (Z-DNA), whereas the unmethylated (dC-dG)(4) analog remains right-handed under those conditions. ITC was then used to determine the enthalpy change upon increasing the concentration of either Na(+) or [Co(NH(3))(6)](3+) for both DNA oligomers at 25 degrees C. The titration with Na(+) resulted in endothermic isotherms with (dm(5)C-dG)(4) being more endothermic than (dC-dG)(4) by 700 cal/mol basepair. In contrast, titration with [Co(NH(3))(6)](3+) resulted in exothermic isotherms with (dC-dG)(4) being more exothermic than (dm(5)C-dG)(4) by 720 cal/mol basepair. We attribute the enthalpy difference to the conformational transition from B-form DNA to Z-form DNA for (dm(5)C-dG)(4), a transition which does not occur for the unmethylated (dC-dG)(4). The value of approximately 700 cal/mol basepair for the enthalpy of the B-Z transition compares favorably with previously published results obtained by different techniques. DSC was used to monitor the duplex to single strand transitions for both oligomers under the different concentrations. These results indicated that methylation of the cytidine destabilizes (dm(5)C-dG)(4) relative to (dC-dG)(4). Coupling the DSC data with the ITC data allowed construction of a thermodynamic cycle which gives insight into the influence of both temperature and ionic strength on the heat content of the two DNA systems studied. Further, this study reveals the utility of using ITC for determinations of transition enthalpies with the appropriate choice of control.     

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.     

Application of isothermal titration calorimetry in bioinorganic chemistry

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC _p°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K _ITC, ΔH _ITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.     

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.     

Thermodynamics of cationic lipid-DNA complex formation as studied by isothermal titration calorimetry

The detailed analysis of the cationic lipid-DNA complex formation by means of isothermal titration calorimetry is presented. Most experiments were done using 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (EDOPC), but basic titrations were also done using DOTAP, DOTAP:DOPC, and DOTAP:DOPE mixtures. Complex formation was endothermic with less than 1 kcal absorbed per mole of lipid or DNA charge. This enthalpy change was attributed to DNA-DNA mutual repulsion within the lamellar complex. The exception was DOTAP:DOPE-containing lipoplex for which the enthalpy of formation was exothermic, presumably because of DOPE amine group protonation. Experimental conditions, namely, direction and titration increment as well as concentration of titrant, which dictate the structure of resulting lipoplex (whether lamellar complex or DNA-coated vesicle), were found to affect the apparent thermodynamics of complex formation. The structure, in turn, influences the biological properties of the lipoplex. If the titration of lipid into DNA was carried out in large increments, the DeltaH was larger than when the injection increments were smaller, a finding that is consistent with increased vesicle disruption under large increments and which is expected theoretically. Cationic lipid-DNA binding was weak in high ionic strength solutions, however, the effective binding constant is within micromolar range because of macromolecular nature of the interaction.     

The use of isothermal titration calorimetry to unravel chemotactic signalling mechanisms

Chemotaxis is based on the action of chemosensory pathways and is typically initiated by the recognition of chemoeffectors at chemoreceptor ligand-binding domains (LBD). Chemosensory signalling is highly complex; aspect that is not only reflected in the intricate interaction between many signalling proteins but also in the fact that bacteria frequently possess multiple chemosensory pathways and often a large number of chemoreceptors, which are mostly of unknown function. We review here the usefulness of isothermal titration calorimetry (ITC) to study this complexity. ITC is the gold standard for studying binding processes due to its precision and sensitivity, as well as its capability to determine simultaneously the association equilibrium constant, enthalpy change and stoichiometry of binding. There is now evidence that members of all major LBD families can be produced as individual recombinant proteins that maintain their ligand-binding properties. High-throughput screening of these proteins using thermal shift assays offer interesting initial information on chemoreceptor ligands, providing the basis for microcalorimetric analyses and microbiological experimentation. ITC has permitted the identification and characterization of many chemoreceptors with novel specificities. This ITC-based approach can also be used to identify signal molecules that stimulate members of other families of sensor proteins.     

Partition of amphiphilic molecules to lipid bilayers by isothermal titration calorimetry

The partition of the amphiphile sodium dodecyl sulfate (SDS) between an aqueous solution and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was followed by isothermal titration calorimetry (ITC) as a function of the total concentration of SDS. It was found that the obtained partition coefficient is strongly affected by the ligand concentration, even after correction for the charge imposed in the bilayer by the bound SDS. The partition coefficient decreased as the total concentration of SDS increased, with this effect being significant for local concentrations of SDS in the lipid bilayer above 5 molar%. At those high local concentrations, the properties of the lipid bilayer are strongly affected, leading to nonideal behavior and concentration-dependent apparent partition coefficients. It is shown that with the modern ITC instruments available, the concentrations of SDS can be drastically reduced while maintaining a good signal-to-noise ratio. The intrinsic parameters of the interaction with unperturbed membranes can be obtained from the asymptotic behavior of the apparent parameters as a function of the ligand concentration for both nonionic and ionic solutes. A detailed analysis is performed, and a spreadsheet is provided to obtain the interaction parameters with and without correction for electrostatics.     

A study of statistical error in isothermal titration calorimetry

In isothermal titration calorimetry (ITC), the two main sources of random (statistical) error are associated with the extraction of the heat q from the measured temperature changes and with the delivery of metered volumes of titrant. The former leads to uncertainty that is approximately constant and the latter to uncertainty that is proportional to q. The role of these errors in the analysis of ITC data by nonlinear least squares is examined for the case of 1:1 binding, M+X right arrow over left arrow MX. The standard errors in the key parameters-the equilibrium constant Ko and the enthalpy DeltaHo-are assessed from the variance-covariance matrix computed for exactly fitting data. Monte Carlo calculations confirm that these "exact" estimates will normally suffice and show further that neglect of weights in the nonlinear fitting can result in significant loss of efficiency. The effects of the titrant volume error are strongly dependent on assumptions about the nature of this error: If it is random in the integral volume instead of the differential volume, correlated least-squares is required for proper analysis, and the parameter standard errors decrease with increasing number of titration steps rather than increase.     

Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics

Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such studies. The power of ITC lies in its unique ability to measure binding reactions by the detection of the heat change during the binding interaction. Since heat changes occur during many physicochemical processes, ITC has a broad application, ranging from chemical and biochemical binding studies to more complex processes involving enthalpy changes, such as enzyme kinetics. Several features of ITC have facilitated its preferential use compared to other techniques that estimate affinity. It is a sensitive, rapid, and direct method with no requirement for chemical modification or immobilization. It is the only technique that directly measures enthalpy of binding and so eliminates the need for van't Hoff analysis, which can be time consuming and prone to uncertainty in parameter values. Although ITC has facilitated the measurement of the thermodynamics governing binding reactions, interpretation of these parameters in structural terms is still a major challenge.     

A "release" protocol for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state.     

Biothermodynamic analysis of BSA adsorption to alum gel using isothermal titration calorimetry

In this study, we used ITC (isothermal titration calorimetry) to quantitatively investigate the impacts of temperature and protein concentration on adsorption behavior on a solid surface, using BSA (bovine serum albumin) as a model protein, and alum (aluminum hydroxide) gel as an adsorbent. The zeta potential measurement for alum gel (0.25 mV at pH 9.3) revealed that its surface charge was not strong enough for electrostatic interaction. ITC analysis showed that the BSA-alum gel interaction was entropy-driven, suggesting that during adsorption, water molecules were expelled from the hydration layers of the alum gel and BSA. Therefore, the major mechanism for the BSA-alum gel interaction was hydrophobic interaction rather than electrostatic interaction. This biothermodynamic approach can be helpful not only to identify interaction mechanisms, but also to explore the optimum conditions for protein-adsorbent interactions.     

Study of inclusion complexes of cycloamylose with surfactants by isothermal titration calorimetry

Useful materials can be made from cycloamylose (CA) and the functional properties of CA could be improved by complexation with surfactants. Isothermal titration calorimetry (ITC) was used to investigate interactions between CA and surfactants in buffered solutions. Three surfactants with C₁₂ non-polar tail groups and charged [anionic: sodium dodecyl sulfate (SDS); cationic: dodecyl trimethylammonium bromide (DTAB)] or non-charged headgroups [non-ionic: polyoxyethylene 23 lauryl ether (Brij35)] were used in this study. The effects of temperature, pH, and salt concentration were also studied. All three surfactants bound to CA; however, Brij35 binding to CA was negligible. Enthalpy changes associated with binding of surfactants to CA were exothermic except for interactions measured at 50 °C. There was no effect of pH on surfactant demicellization or CA binding. Salt concentration affected surfactant demicellization, but the amount of SDS bound to CA at saturation was unaffected by salt. When the titration curves obtained for CA with SDS and DTAB were fitted, it could be analyzed using a model based on a single set of identical sites.     

Applications of isothermal titration calorimetry in RNA biochemistry and biophysics

Isothermal titration calorimetry (ITC) has been applied to the study of proteins for many years. Its use in the biophysical analysis of RNAs has lagged significantly behind its use in protein biochemistry, however, in part because of the relatively large samples required. As the instrumentation has become more sensitive, the ability to obtain high quality data on RNA folding and RNA ligand interactions has improved dramatically. This review provides an overview of the ITC experiment and describes recent work on RNA systems that have taken advantage of its versatility for the study of small molecule binding, protein binding, and the analysis of RNA folding.