Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (k_cat, K_M, ΔH) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine k_cat and K_M of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (ΔH_int) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.     

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.     

Thermodynamic analyses reveal role of water release in epitope recognition by a monoclonal antibody against the human guanylyl cyclase C receptor.

The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.     

Stoichiometry of the reactions of calcium with the metallochromic indicator dyes antipyrylazo III and arsenazo III.

A method for determining the stoichiometry of one-product reactions involving a metal ion and an organic ligand is presented and applied to the reactions of calcium and magnesium with the metallochromic dyes Antipyrylazo III and Arsenazo III. The method consists of fitting titration data, obtained in solutions buffered for the metal, with theoretical functions that include: (a) the dependence of product concentration on the concentration of both reactants, (b) the relationship between metal ion concentration and total amount added in the presence of the buffer, and (c) a correction for the amount of metal ion that binds to the organic ligand. It is shown that the products of the reactions of Antipyrylazo III with calcium and magnesium are CaD2 and MgD, respectively. The product formed between calcium and Arsenazo III at [Ca2+] over 20 microM is CaD2, other products accumulating at lower [Ca2+]. The kinetics of the Antipyrylazo III:Ca reaction are rapid under conditions in which this dye has been applied to measure calcium transients in skeletal muscle fibers. The present results provide a calibration for previous studies with Antipyrylazo III in muscle fibers.     

A twin titration microcalorimeter for the study of biochemical reactions

A small-volume (200 microliter) titration calorimeter of high sensitivity (1 mu cal ) has been developed for the purpose of studying biochemical reactions where the amounts of material are limited to a few nanomoles. High sensitivity is achieved by calorimetric twining , use of glass cells, elimination of vapor space, effective low-energy stirring, and reduction of measurement time. The calorimeter operates using the heat conduction principal with computer-controlled electrical compensation, which reduces the measurement time of each point from 10 to 3 min. This reduction in time is accompanied by a corresponding increase in the precision of measurement. The use of the calorimeter is demonstrated by a measurement of the heat of oxygenation of hemocyanin.     

Use of the twin-cell differential titration calorimeter for binding studies. I. EDTA and its calcium complex

The use of a twin-cell differential titration calorimeter is described which utilizes small volumes (1–3 ml) of modest concentrations of materials (0.001–0.01 M) and yield data of good precision. Operation is controlled by a microprocessor which regulates and controls the addition of reagents and collects and displays the data as time, temperature in volts, and the pH. Corrections for the titration of water are applied to the potentiometric data, and the thermal data are corrected for the initial temperature-time baseline, the changes in heat capacity, and the heat loss (or gain) to the external environment. Finally, the thermal signal is corrected for the heat derived from the formation of water due to the free hydrogen or hydroxyl ions present. The corrected data as pH, groups titrated adn ΔH_T (kcal/mol) can then be used to obtain the parameters pK′ and ΔH_i involved with the equilibria by curve-fitting the observed data.

The system has been applied to the ionization of EDTA and its calcium complex. The ionization constants, the heats of ionization, the stoichiometry of binding and the heat of binding have been determined and demonstrated to be in agreement with published values.     

Estimations on the degradability of ores and bacterial leaching activity using short-time microcalorimetric tests

Abstract: This work is based on calorimetric measurements on batch cultures of ferrous iron-oxidizing thiobacilli and leaching cultures degrading solid substrates in the titration vessel under aerobic conditions. It has been demonstrated that heat-flow levels are a direct measure for oxidation rates of known reactions under non-limiting conditions. The total loss of heat-energy per unit of ferrous iron oxidized is identical under similar incubation conditions with different Thiobacillus ferrooxidans strains and different cell-densities in culture suspension. Under limiting conditions a reduced heat-loss occurred. This may have been due to either a more effective use of the substrate or to a shift to different redox reactions. Oxygen was proved to be a limiting factor in dense bacterial cultures with high substrate concentrations in the titration vessel. The experimental duration decreased significantly with the use of increasing cell densities. Therefore cell densities in degradation experiments with solid substrates were adjusted to between 5X10⁹ and 1x10¹⁰ cells/ml in short-term experiments. T. ferrooxidans is the most important species for short-term leaching experiments. Calorimetric measurements allow the best strains for a degradation of unknown substrates to be found. The calculated heat energy is a measure of the amount of converted substrate. High heat-flow only occurs if catabolic reactions take place and maintenance reactions and phenomena like adhesion of cells to surface do not cause significant loss of heat.     

Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.     

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.     

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

Survey of the year 2008: applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast, accurate and label‐free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes. Not only binding thermodynamics but also catalytic reactions, conformational rearrangements, changes in protonation and molecular dissociations can be readily quantified by performing only a small number of ITC experiments. In this review, we highlight some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs. As is tradition in these reviews we have not attempted a comprehensive analysis of all 500 papers using ITC, but emphasize those reports that particularly captured our interest and that included more thorough discussions we consider exemplify the power of the technique and might serve to inspire other users. Copyright © 2010 John Wiley & Sons, Ltd.     

Reverse engineering of biochemical equations from time-course data by means of genetic programming

Increased research aimed at simulating biological systems requires sophisticated parameter estimation methods. All current approaches, including genetic algorithms, need pre-existing equations to be functional. A generalized approach to predict not only parameters but also biochemical equations from only observable time-course information must be developed and a computational method to generate arbitrary equations without knowledge of biochemical reaction mechanisms must be developed. We present a technique to predict an equation using genetic programming. Our technique can search topology and numerical parameters of mathematical expression simultaneously. To improve the search ability of numeric constants, we added numeric mutation to the conventional procedure. As case studies, we predicted two equations of enzyme-catalyzed reactions regarding adenylate kinase and phosphofructokinase. Our numerical experimental results showed that our approach could obtain correct topology and parameters that were close to the originals. The mean errors between given and simulation-predicted time-courses were 1.6 x 10(-5)% and 2.0 x 10(-3)%, respectively. Our equation prediction approach can be applied to identify metabolic reactions from observable time-courses.     

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.     

An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

A survey of the year 2006 literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast and robust method to determine the energetics of association reactions in solution. The changes in enthalpy, entropy and heat capacity that accompany binding provide unique insights into the balance of forces driving association of molecular entities. ITC is used nowadays on a day-to-day basis in hundreds of laboratories. The method aids projects both in basic and practice-oriented research ranging from medicine and biochemistry to physical chemistry and material sciences. Not surprisingly, the range of studies utilizing ITC data is steadily expanding. In this review, we discuss selected results and ideas that have accumulated in the course of the year 2006, the focus being on biologically relevant systems. Theoretical developments, novel applications and studies that provide a deeper level of understanding of the energetic principles of biological function are primarily considered. Following the appearance of a new generation of titration calorimeters, recent papers provide instructive examples of the synergy between energetic and structural approaches in biomedical and biotechnological research.     

Phytic acid-zinc ion interactions: a calorimetric and titrimetric study

Using an isoperibolic titration microcalorimeter, the ionization characteristics and associated heat changes of phytic acid (myo-inositol hexaphosphate) and phytic acid in the presence of varying Zn(II) concentrations have been examined over the pH range 2.5–11 at 25°C in 0.2 M KCl. In the absence of Zn(II), ca. 7 of the 12 ionizable protons in phytic acid are titrated in this pH range with ionization heats varying from ca. 2 to −3 kcal-mol^-1. At Zn(II): phytate mol ratios of 4:1 and greater, the dissociation of all protons and complex formation of phytate with Zn(II) occurs below pH 6. From the difference titration curves of phytic acid plus Zn(II) versus Zn(II) alone, ca. 3.5 mol Zn(II) bind per mol phytate. Since Zn(II):phytate complexes are insoluble, the observed heat changes contain contributions not only from heats of precipitation but also from binding, ionization, neutralization, and hydration effects. From the heat change for the titration of (a) phytic acid, pH 2.6–10.4; (b) phytic acid + Zn(II), pH 2.6–6.1; and (c) Zn(II), pH 2.6–6.1 at Zn(II): phytate ratios of 4 to 10, the value of 24.7 ± 0.5 kcal mol⁻¹ phytate has been obtained for the binding of 3.5 mols Zn(II). This figure also includes the heat of precipitation of the complex. In pH-drop experiments, with the initial pH at 8.65, the value of 23.9 kcal mol^-1 was obtained for ΔH°. Hysteresis effects are prevalent in these reaction solutions. Time-dependent changes in pH occur with a change in pH. For the phytate-Zn(II) reactions, the time-course curves are biphasic and fit a rate equation for two simultaneous first order reactions. Hysteresis effects seen in the titration of Zn(II) fit simple first-order kinetics. These effects most probably arise from the ejection of a proton from the aqua ion or aqua ion ligand complex(es).     

Thermodynamic investigations of protein's behaviour with ionic liquids in aqueous medium studied by isothermal titration calorimetry

BackgroundWhile a number of reports appear on ionic liquids–proteins interactions, their thermodynamic behaviour using suitable technique like isothermal titration calorimetry is not systematically presented.MethodsIsothermal titration calorimetry (ITC) is a key technique which can directly measure the thermodynamic contribution of IL binding to protein, particularly the enthalpy, heat capacities and binding stoichiometry.Scope of reviewIonic liquids (ILs), owing to their unique and tunable physicochemical properties have been the central area of scientific research besides graphene in the last decade, and growing unabated. Their encounter with proteins in the biological system is inevitable considering their environmental discharge though most of them are recyclable for a number of cycles. In this article we will cover the thermodynamics of proteins upon interaction with ILs as osmolyte and surfactant. The up to date literature survey of IL–protein interactions using isothermal titration calorimetry will be discussed and parallel comparison with the results obtained for such studies with other techniques will be highlighted to demonstrate the accuracy of ITC technique.Major conclusions and general significanceNet stability of proteins can be obtained from the difference in the free energy (ΔG) of the native (folded) and denatured (unfolded) state using the Gibbs–Helmholtz equation (ΔG = ΔH − TΔS). Isothermal titration calorimetry can directly measure the heat changes upon IL–protein interactions. Calculation of other thermodynamic parameters such as entropy, binding constant and free energy depends upon the proper fitting of the binding isotherms using various fitting models. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Hydration changes in the association of Hoechst 33258 with DNA

The role of water in the interaction of Hoechst 33258 with the minor groove binding site of the (AATT)2 sequence was investigated using calorimetric and equilibrium constant measurements. Using isothermal titration calorimetry measurements, the heat capacity change for the reaction is -256 +/- 10 cal/(K mol of Hoechst). Comparison with the heat capacity changes based on area models supports the expulsion of water from the interface of the Hoechst-DNA complex. To further consider the role of water, the osmotic stress method was used to determine if the Hoechst association with DNA was coupled with hydration changes. Using four osmolytes with varying molecular weights and chemical properties, the Hoechst affinity for DNA decreases with increasing osmolyte concentration. From the dependence of the equilibrium constant on the solution osmolality, 60 +/- 13 waters are acquired in the complex relative to the reactants. It is proposed that the osmotic stress technique is measuring weakly bound waters that are not measured via the heat capacity changes.     

狂犬病毒蚀斑方法在病毒研究和疫苗研制中的应用

本文采用狂犬病毒CTN-1和4aC株,经Vero细胞传代适应后,以Vero细胞为培养基质,建立了狂犬病毒蚀斑试验和蚀斑减少试验的方法。目前已将此方法应用于病毒鉴定、病毒克隆、病毒滴定以及抗狂犬血清的检测,并取得了较好的结果。