A review on the ligand binding studies by isothermal titration calorimetry

Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (ΔG), the enthalpy of binding (ΔH) and the entropy of binding (ΔS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.     

Thermodynamics of protein denaturation by sodium dodecyl sulfate

The anionic surfactant sodium n-dodecyl sulfate (SDS) plays a variety of roles with regard to protein conformation, depending on its concentration. SDS at low concentrations mostly induces the compaction of protein (folding). Examples of this include: the molten globule state of acid-unfolded cytochrome c, associated with enhancement of the exothermic enthalpy values of isothermal titration calorimetry and a reversible profile by differential scanning calorimetry; the enzyme activation and compaction of Aspergillus niger catalase, and relationship of calorimetric enthalpy (ΔH_cal) to van’t Hoff enthalpy (ΔH_VH), which proves the existence of intermolecular and intramolecular interaction during enzyme activation by SDS; the production of a new energetic domain for human apotransferrin and folded state for histone H1 by SDS. SDS at moderate concentrations below the critical micelle concentration (cmc) is a potent denaturant for protein in solution. Protein denaturation is a key method in thermodynamics and binding site analysis and can be used to enhance our understanding of the protein structure-function relationship. The interaction between protein and surfactant, such as SDS, at the cmc level is a complicated interaction, thermodynamically, that should bring about enthalpy correction through micellar dissociation and micelle dilution.     

On the partial reversibility of the Β-lactoglobulin heat denaturation

The thermal behavior ofΒ-lactoglobulin (Β-lg) disperesed in distilled water (pH=3.2) is studied dy differential scanning calorimetry (DSC) in the temperature range 20?C–120?C and within a concentration region of 3.5% to 24%. Recently [1] we have determined by DSC the kinetic parameters for the heat-denaturation ofΒ-lg. The effect of the protein concentration and of the thermal treatment on transition temperature (T _trs), half-widths of the peak (δT _1/2), of apparent enthalpy changes (δ_app H) and of Van't Hoff enthalpies (δ_VH H) have been examined for the concentrations of 8.8% and 24%. In this study we have undertaken complementary experiments for the concentrations 3.5% and 10.8%. The half-widths of the peaks, which depend on the cooperativity of the denaturation process [2], decrease with increasing concentration. The ratio δ_app H/δ_VH H tends to 1, with low values of the protein concentration and with high scanning rates. This implies the hypothesis of a reversible step for the denaturation process ofΒ-lg.     

Hydrophobic Effect: Solubility of Non-polar Substances in Water,Protein Denaturation and Micelle Formation

The 'hydrophobic effect' of the dissolution process of non-polar substances in water has been analysed under the light of a statistical thermodynamic molecular model. The model, based on the distinction between non-reacting and reacting systems explains the changes of the thermodynamic functions with temperature in aqueous systems. In the dissolution of non-polar substances in water, it follows from the model that the enthalpy change can be expressed as a linear function of the temperature (ΔH _app =ΔH ^ø +n _w C _p,w T ). Experimental solubility and calorimetric data of a large number of non-polar substances nicely agree with the expected function. The specific contribution of n _w solvent molecules depends on the size of solute and is related to destructuring (n _w >0) of water molecules around the solute. Then the study of 'hydrophobic effect' has been extended to the protein denaturation and micelle formation. Denaturation enthalpy either obtained by van't Hoff equation or by calorimetric determinations again depends linearly upon denaturation temperature, with denaturation enthalpy, ΔH _den , increasing with T . A portion of reaction enthalpy is absorbed by a number n _w of water molecules (n _w >0) relaxed in space around the denatured moieties. In micellization, an opposite process takes place with negative number of restructured water molecules (n _w <0) because the hydrophobic moieties of the molecules joined by hydrophobic affinity occupy a smaller cavity.     

Dissolution properties of N-guanylurea dinitramide (GUDN) in dimethyl sulfoxide and N-methyl pyrrolidone

The enthalpies of dissolution of N-guanylurea dinitramide (GUDN) in dimethyl sulfoxide (DMSO) and N-methyl-2-pyrrolidone (NMP) were measured using an RD496-2000 Calvet microcalorimeter at 298.15 K under atmospheric pressure, respectively. Empirical formulae for the calculation of the enthalpy of dissolution (Δ_diss H), relative partial molar enthalpy (Δ_diss H _partial), and relative apparent molar enthalpy (Δ_diss H _apparent) were obtained from the experimental data of the dissolution processes of GUDN in DMSO and NMP. Furthermore, the corresponding kinetic equations describing the two dissolution processes were dα/dt = 10^?3.39(1 ? α)^0.70 for the dissolution of GUDN in DMSO, and dα/dt = 10^?4.06(1 ? α)^1.11 for the dissolution of GUDN in NMP.     

Thermal stability of lysozyme and myoglobin in the presence of anionic surfactants

The interactions of lysozyme and myoglobin with anionic surfactants (hydrogenated and fluorinated), at surfactant concentrations below the critical micelle concentration, in aqueous solution were studied using spectroscopic techniques. The temperature conformational transition of globular proteins by anionic surfactants was analysed as a function of denaturant concentration through absorbance measurements at 280 nm. Changes in absorbance of protein-surfactant system with temperature were used to determine the unfolding thermodynamics parameters, melting temperature, T _m, enthalpy, ΔH _m, entropy, ΔS _m and the heat capacity change, ΔC _p, between the native and denatured states.     

Effect of the composition of a water-alcohol solvent on the thermodynamics of dissolution of DL-α-alanyl-β-alanine at 298.15 K

Enthalpies of solution for DL-α-alanyl-β-alanine in H₂O-ethanol, H₂O-1-propanol, and H₂O-2-propanol mixed solvents with the alcohol mole fraction x ₂ = 0–0.3 are measured at 298.15 K. Standard enthalpies of solution (Δ_sol H ^°), standard enthalpies of transfer of DL-α-alanyl-β-alanine from water to binary solvent (Δ_tr H ^°), and coefficients of enthalpies of pair interactions with alcohol molecules (h _xy ) are calculated. The effect the structure and properties of alcohols and the composition of a water-alcohol mixture have on the enthalpy of dissolution for DL-α-alanyl-β-alanine are discussed. The h _xy values for dipeptides of the alanine series in water-alcohol binary solvents are compared.     

Effect of venous wall immobilization on the thermal degradation of collagen

The results from a comparative study of the thermal denaturation of collagen in the venous walls of reference samples and samples with varicose disease are presented. Changes in the organization of collagen network of the tissue matrix are detected via thermal analysis and multiphoton microscopy with recording of the second harmonic generation (SHG). It is established that the collagen network of venous walls degrades in varicose disease. It is shown that the disordering of the tertiary structure of collagen molecules is reflected in a 40% drop in the enthalpy of protein denaturation compared to reference (ΔH _D = 12.4 ± 4.9 J/g dry residue). The disorganization of fiber structures is recorded on SHG images. It is shown that upon the hydrothermal heating of sequestered samples of venous walls, the complete degradation of the tissue network occurs at 75°C. However, it is noted that upon the mechanical immobilization of samples of both types, the stability of collagen increases and complete denaturation is observed at temperatures above 84°C. It is suggested that the number of available conformations of polypeptide chains in the random coil state falls under tension, lowering ΔS _D and raising the temperature of the denaturation of protein.     

The adsorption of phosphamidon on the surface of antimony(V) phosphate: A thermodynamic study

The thermodynamics of the adsorption of phosphamidon on antimony(V) phosphate cation exchanger has been studied at 30, 45 and 50°C and the thermodynamic equilibrium constant (K₀), standard free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) have been calculated to predict its adsorption behaviour. All the data are adequately represented by the Freundlich isotherms.     

Conformational and phase transitions in lysozyme-thermosensitive polyelectrolyte complexes

The interaction of a small globular protein, lysozyme, with a thermosensitive N-isopropylacrylamide-sodium styrene sulfonate copolymer at pH 4.6 was studied by high-sensitivity differential scanning calorimetry. It was shown that, under these conditions, the copolymer and the protein are involved in formation of polyelectrolyte complexes. It was demonstrated that complexation affects the conformational state of lysozyme. One heat capacity peak attributed to protein denaturation or two well-resolved peaks related to denaturation of free and bound proteins were observed in the DSC curves depending on the mixture composition. For both forms of lysozyme, denaturation parameters (temperature and enthalpy) were determined as a function of mixture composition. For mixtures with low lysozyme contents, the above parameters of bound protein denaturation were independent of the mixture composition. At higher protein contents, these parameters increased with a rise in the protein content. The binding isotherm for the protein with the copolymer was obtained from calorimetric data at 64 ± 1°C. An analysis of the isotherm suggests that the native protein is bound to 24 equivalent binding sites of the polymer matrix. It was established that there are nearly 10 charged units of the copolymer per protein molecule in the native conformation. Lysozyme in the unfolded conformation additionally interacts with hydrophobic groups of the copolymer.     

Surface Characterization of Sepiolite by Inverse Gas Chromatography

Inverse gas chromatography (IGC) was applied to characterize the surface of sepiolite. The adsorption thermodynamic parameters (the standard enthalpy (ΔH ⁰), entropy (ΔS ⁰) and free energy of adsorption (ΔG ⁰)), the dispersive component of the surface energy (γ _S ^d ), and the acid/base character of sepiolite surface were estimated by using the retention time of different non-polar and polar probes at infinite dilution region. The specific free energy of adsorption (ΔG ^sp ), the specific enthalpy of adsorption (ΔH ^sp ), and the specific entropy of adsorption (ΔS ^sp ) of polar probes on sepiolite were determined. ΔH ^sp were correlated with the donor and modified acceptor numbers of the probes to quantify the acidic K _A and the basic K _D parameters of the sepiolite surface. The values obtained for the parameters K _A and K _D indicated an acidic character for sepiolite surface.     

The solvation of L-serine in mixtures of water with some aprotic solvents at 298.15 K

The integral enthalpies of solution Δ_sol H ^m of L-serine in mixtures of water with acetonitrile, 1,4-dioxane, dimethylsulfoxide (DMSO), and acetone were measured by solution calorimetry at organic component concentrations up to 0.31 mole fractions. The standard enthalpies of solution (Δ_sol H°), transfer (Δ_tr H°), and solvation (Δ_solv H°) of L-serine from water into mixed solvents were calculated. The dependences of Δ_sol H°, Δ_solv H°, and Δ_tr H° on the composition of aqueous-organic solvents contained extrema. The calculated enthalpy coefficients of pair interactions of the amino acid with cosolvent molecules were positive and increased in the series acetonitrile, 1,4-dioxane, DMSO, acetone. The results obtained were interpreted from the point of view of various types of interactions in solutions and the influence of the nature of organic solvents on the thermochemical characteristics of solutions.     

Optical Spectroscopic Studies on Structural Changes of Helical-rich Proteins in Aqueous Solutions of Ionic Liquids

We have investigated structural changes of myoglobin and cytochrome c, which are helical-rich proteins, in aqueous 1-butyl-3-methylimidazolium chloride ([bmim][Cl]) solutions by Fourier transform infrared and circular dichroism spectroscopy. At low [bmim][Cl] concentrations {X (mol% IL) < 10}, both proteins unfold. Remarkably, at high [bmim][Cl] concentrations (X > 10), myoglobin aggregates whereas cytochrome c refolds its α-helical structure. The tertiary structures of both proteins are disrupted over the entire range of studied [bmim][Cl] concentrations. Our results suggest that, in aqueous solutions at high [bmim][Cl] concentrations, the differences in structural transitions between myoglobin and cytochrome c might be due to the difference in hydration between these proteins.     

Relationship between the molecular structure of amino acids and dipeptides and thermal sublimation effects

This paper reports on our mass spectrometric study of sublimation of glycine and DL-alanyl-glycine (Ala-gly). The sublimation enthalpy of Ala-gly has been determined by generalization of the data obtained and the results of AM1 quantum-chemical calculations. A relationship has been found between the sublimation enthalpy (ΔH _subl), heat capacity (C _P), and the sum of bond lengths (Σn _i l _i ) in 17 α-amino acid and 9 dipeptide molecules. Correlations are suggested for evaluating ΔH _subl of amino acids and peptides.     

A Comparative Study of the Direct Calorimetric Determination of the Denaturation Enthalpy for Lysozyme in Sodium Dodecyl Sulfate and Dodecyltrimethylammonium Bromide Solutions

The complexes of lysozyme with the anionic surfactant sodium dodecyl sulfate (SDS) and the cationic surfactant dodecyltrimethylammonium bromide (DTAB) have been investigated by isothermal titration calorimetry at pH=7.0 and 27 °C in a phosphate buffer. A new direct calorimetric method was applied to follow the protein denaturation and study the effect of surfactants on the stability of proteins. The extended solvation model was used to represent the enthalpies of lysozyme + SDS interaction over the whole range of SDS concentrations. The solvation parameters recovered from the new equation are attributed to the structural change of lysozyme and its biological activity. At low SDS concentrations, the binding is mainly electrostatic with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic regions of lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The induced enthalpy of denaturation of lysozyme by SDS is 160.81±0.02 kJ⋅mol⁻¹. The lysozyme-DTAB complexes behave very differently from those of the lysozyme-SDS complexes. SDS induces a stronger unfolding of lysozyme than DTAB. The induced enthalpy of lysozyme denaturation by DTAB is 86.46±0.02 kJ⋅mol⁻¹.     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.     

Effect of enthalpy of polar modifiers interaction with n-butyllithium on the reaction enthalpy,kinetics and chain microstructure during anionic polymerization of 1,3-butadiene

The influence of interaction enthalpy (ΔH_MOD/BuLi) of μ, σ, σ+μ and σ-μ complexing polar modifiers with n-butyllithium on the 1,3-butadiene anionic polymerization enthalpy (ΔH_BD), polymerization reaction rate (k_p) and polybutadiene microstructure was studied. It has been found that enthalpy of interaction depends on complex type, molar ratio of polar modifier to n-butyllithium (MOD/BuLi) and temperature. For the first time it has been proven that with increasing ΔH_MOD/BuLi the content of 1,2 butadiene (vinyl) units in the chain increases individually for each complex type but the value of ΔH_BD decreases similarly for all complexes containing σ-donor groups, exhibiting linear nature. Since ΔH_MOD/BuLi controls the content of butadiene isomeric structures in the chain its value was compared with polymerization reaction rate, ranging from ∼46 to ∼5500 moL/L·min, and discussed on mechanistic level.     

The standard enthalpies of formation of cyclohexylamine and cyclohexylamine hydrochloride

The standard enthalpy of combustion of cyclohexylamine has been measured in an aneroid rotating-bomb calorimeter. The value ΔH_o^o(c-C₆H₁₁NH₂, 1) = ?(4071.3 ± 1.3) kJ mol^?1 yields the standard enthalpy of formation ΔH_f^o(c-C₆H₁₁NH₂, 1) = ?(147.7 ± 1.3) kJ mol^?1. The corresponding gas-phase standard enthalpy of formation for cyclohexylamine is ΔH_f^o(c-C₆H₁₁NH₂, g) = ?(104.9 ± 1.3) kJ mol^?1. The standard enthalpy of formation of cyclohexylamine hydrochloride, ΔH_f^o(c-C₆H₁₁NH₂·HCl, c) = ?(408.2 ± 1.5) kJ mol^?1, was derived by combining the measured enthalpy of solution of the salt in water, literature data, and the ΔH_c^o measured in this study. Comment is made on the thermochemical bond enthalpy H(CN).     

Molar heat capacities and standard molar enthalpy of formation of pyrimethanil butanedioic salt

A noval anilino-pyrimidine fungicide, pyrimethanil butanedioic salt (C₂₈H₃₂N₆O₄), was synthesized by a chemical reaction of pyrimethanil and butanedioic acid. The low-temperature heat capacities of the compound were measured with an adiabatic calorimeter from 80 to 380 K. The thermodynamic function data relative to 298.15 K were calculated based on the heat capacity fitted curve. The thermal stability of the compound was investigated by TG and DSC. The TG curve shows that pyrimethanil butanedioic salt starts to sublimate at 455.1 K and totally changes into vapor when the temperature reaches 542.5 K with the maximal speed of weight loss at 536.8 K. The melting point, the molar enthalpy (Δ_fus H _m), and entropy (Δ_fus S _m) of fusion were determined from its DSC curves. The constant-volume energy of combustion (Δ_c U _m) of pyrimethanil butanedioic salt was measured by an isoperibol oxygen-bomb combustion calorimeter at T = (298.15 ± 0.001) K. From the Hess thermochemical cycle, the standard molar enthalpy of formation was derived and determined to be Δ_f H _m ^o (pyrimethanil butanedioic salt)=?285.4 ± 5.5 kJ mol^?1.     

Effects of water-alcohol binary solvents on the thermochemical characteristics of L-tryptophane dissolution at 298.15 K

The enthalpies of L-tryptophane solution in water-methanol, water-ethanol, water-1-propanol, and water-2-propanol mixtures at alcohol concentrations of x ₂ = 0–0.4 mole fractions were measured by calorimetry. The standard enthalpies of L-tryptophane solution (Δ_sol H ^°) and transfer (Δ_tr H ^°) from water to the binary solvent were calculated. The influence of the composition of the water-alcohol mixture and the structure and properties of L-tryptophane on the enthalpy characteristics of the latter was considered. The enthalpy coefficients of pair interactions (h _xy ) of L-tryptophane with alcohol molecules were calculated. The coefficients were positive and increased in the series: methanol (MeOH), ethanol (EtOH), 1-propanol (1-PrOH), and 2-propanol (2-PrOH). The solution and transfer enthalpies of L-tryptophane were compared with those of aliphatic amino acids (glycine, L-threonine, DL-alanine, L-valine, and L-phenylalanine) in similar binary solvents.