A convenient tool for studying the stability of proteins and nucleic acids

The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.     

Thermal stability of porcine pepsin influenced by Al(III) ion: DSC study

Differential scanning calorimetry (DCS) has been used to determine thermodynamic profile of pepsin and the in vitro effect of Al(III) ions. Thermograms of pepsin unfolding in the presence and absence of aluminum were used to determine the binding constant, K _L, in the pepsin-aluminium model system. The thermodynamic parameters were derived from DSC profiles at different ligand concentrations (1, 5 and 10 mM). The temperatures of thermal transitions (T _m), calorimetric (ΔH ^cal) and van’t Hoff enthalpy (ΔH ^VH), Gibbs free energy, Δ(ΔG), of Al(III) binding to pepsin, as well as an average number of ligands bound to the native protein, were obtained from DSC profiles too. Temperature-dependent changes in the protein structure were also monitored by native PAGE electrophoresis. Increasing the temperature causes the decrease in electrophoretic mobility. Increase in concentration of Al(III) decelerate the migration of pepsin samples on concentration dependent manner. Analysis showed that ligand binding increases thermal stability of protein.     

Scan-rate-dependent melting transitions of interleukin-1 receptor (type II): elucidation of meaningful thermodynamic and kinetic parameters of aggregation acquired from DSC simulations

The role of thermal unfolding as it pertains to thermodynamic properties of proteins and their stability has been the subject of study for more than 50 years. Moreover, exactly how the unfolding properties of a given protein system may influence the kinetics of aggregation has not been fully characterized. In the study of recombinant human Interleukin-1 receptor type II (rhuIL-1R(II)) aggregation, data obtained from size exclusion chromatography and differential scanning calorimetry (DSC) were used to model the thermodynamic and kinetic properties of irreversible denaturation. A break from linearity in the initial aggregation rates as a function of 1/T was observed in the vicinity of the melting transition temperature (T(m) approximately 53.5 degrees C), suggesting significant involvement of protein unfolding in the reaction pathway. A scan-rate dependence in the DSC experiment testifies to the nonequilibrium influences of the aggregation process. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from an irreversibly denatured process. The model was used to simulate how unfolding properties could be used to predict aggregation rates at different temperatures above and below the T(m) and to account for concentration dependence of reaction rates. The model was shown to uniquely identify the thermodynamic parameters DeltaC(P) (1.3 +/- 0.7 kcal/mol-K), DeltaH(m) (74.3 +/- 6.8 kcal/mol), and T(m) with reasonable variances.     

Interchain electron donor–acceptor complexes. Determination of equilibrium constant and thermodynamic parameters in the solid state

The interpolymeric electron donor–acceptor (EDA) complex of donor poly[(N-ethylcarbazol-3-yl)methyl methacrylate] (PHMCM-2) with acceptor poly-(2-[(3,5-dinitrobenzoyl)oxy]ethyl methacrylate) (PDNBM-2) presents a single glass transition temperature and a decomplexation endotherm on differential scanning calorimetric (DSC) thermograms. This system is considered a “polymer blend model” which exhibits a lower critical solution temperature (LCST). Phase separation of this blend is kinetically controlled and positive deviations of the glass transition temperatures from weight average values suggest that it behaves as a thermally reversible crosslinked network. Calorimetric methods to determine the heats of mixing of small molecule complexes in solution were adapted for this solid state blend to estimate the equilibrium constant (K_eq) and other thermodynamic parameters. Applying a computer iterative procedure and assuming 1 : 1 stoichiometry, a least-squares fit was found for several different donor molecular weights with three different high molecular weight acceptors. At moderate molecular weights, K_eq rises to represent saturation fractions near unity as found in biological systems. K_eq decreases for higher molecular weights, possibly due to trapped chain entanglements. These results are supported by a composition-independent, “horizontal line” phase diagram, thus resembling the completely complexed/denaturation process in DNA.     

THERM: Thermodynamic property estimation for gas phase radicals and molecules

We have developed a computer code for an IBM PC/XT/AT or compatible which can be used to estimate, edit, or enter thermodynamic property data for gas phase radicals and molecules using Benson's group additivity method. The computer code is called THERM (THermo Estimation for Radicals and Molecules). All group contributions considered for a species are recorded and thermodynamic properties are generated in old NASA polynomial format for compatibility with the CHEMKIN reaction modeling code. In addition, listings are created in a format more convenient for thermodynamic, kinetic, and equilibrium calculations. Polynomial coefficients are valid from 300–5000 K using extrapolation methods based upon the harmonic oscillator model, an exponential function, or the Wilhoit polynomials. Properties for radical and biradical species are calculated by applying bond dissociation increments to a stable parent molecule to reflect loss of H atom. THERM contains a chemical reaction interpreter to calculate thermodynamic property changes for chemical reactions as functions of temperature. These include equilibrium constant, heat release (required heat, ΔH_r), entropy change (ΔS_r), Gibbs free energy change (ΔG_r), and the ratio of forward to reverse Arrhenius A-factors (for elementary reactions). This interpreter can also process CHEMKIN input files. A recalculation procedure is incorporated for rapid updating of a database of chemical species to reflect changes in estimated bond dissociation energies, heats of formation, or other group values. All input and output files are in ASCII so that they can be easily edited, expanded, or updated.     

Fast method for the experimental determination of vaporization enthalpy by differential scanning calorimetry

A simple method is proposed to estimate the vaporization enthalpy of the palmitic acid (hexadecanoic acid) at its normal boiling temperature. Differential scanning calorimetry (DSC) was the technique used to directly measure these thermodynamic properties. The advantages of this method are its speed and small amount of sample required. In order to avoid evaporation and to ensure equilibrium conditions, the experiments were carried out including a-alumina in contact with the fatty acid. The effect of the alumina concentration is discussed. The obtained experimental data (T_bp=625.4±0.5 K, D_vap H=237.6±5.9 J g^-1) is compared with that obtained by using thermodynamic equations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

An Aqueous Thermodynamic Model for the Complexation of Sodium and Strontium with Organic Chelators Valid to High Ionic Strength. II. N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid (HEDTA)

This is the second paper in a two part series on the development of aqueous thermodynamic models for the complexation of Na⁺ and Sr²⁺ with organic chelators. In this paper, the development of an aqueous thermodynamic model describing the effects of ionic strength, carbonate concentration, and temperature on the complexation of Sr²⁺ by HEDTA under basic conditions is presented. The thermodynamic model describing the Na⁺ interactions with the HEDTA^3–chelate relies solely on the use of Pitzer ion-interaction parameters. The exclusive use of Pitzer ion-interaction parameters differs significantly from our previous model for EDTA, which required the introduction of a NaEDTA^3– ion pair. Estimation of the Pitzer ion-interaction parameters for HEDTA^3– and SrHEDTA^– with Na⁺ allows the extrapolation of a standard-state equilibrium constant for the SrHEDTA^– species, which is one order of magnitude greater than the 0.1 M reference state value available in the literature. The overall model is developed from data available in the literature on apparent equilibrium constants for HEDTA protonation, the solubility of salts in concentrated HEDTA solutions, and from new data on the solubility of SrCO₃(c) obtained as part of this study. The predictions of the final thermodynamic model for the Na-Sr-OH-CO₃-NO₃-HEDTA-H₂O system are tested by application to chemical systems containing competing metal ions (i.e., Ca²⁺).     

Calorimetric and spectroscopic studies characterization of newborn rat’ blood serum after maternal administration of cyclophosphamide

Differential scanning microcalorimetry (DSC) and UV–VIS absorption spectroscopy were used to obtain the characteristics of blood serum from newborn rat’ after maternal treatment with cyclophosphamide in comparison with control. The obtained DSC curves reveal a complex endothermic peak due to the unfolding process of various serum proteins. Thermal profiles and absorption spectra of blood serum are sensitive to the age of newborns as well as to effect of maternal administration of cyclophosphamide. The most significant disturbances in serum proteome were observed for 14-day old newborns. The thermodynamic parameters: enthalpy change (∆H), the normalized first moment (M₁) of the thermal transition with respect to the temperature axis and the ratio of C _p^ex at 70 and 60 °C describing denaturation contributions of globulin forms in respect to unliganded albumin with haptoglobin was estimated. Moreover, the second derivative spectroscopy in the UV region was used to resolve the complex protein spectrum. The differences in blood serum detected by DSC and UV–VIS confirm a potential usefulness of these methods for diagnostic and monitoring changes with age as well as the pathological state of blood serum.     

Thermal denaturation of collagen analyzed by isoconversional method

An isoconversional method is proposed to be used for evaluating activation energy of protein denaturation. Applied to DSC data on collagen denaturation, the method yields an activation energy that decreases throughout the process. The Lumry-Eyring model gives an explanation for this decrease and affords estimates for the enthalpy of the reversible step and the activation energy of the irreversible step of denaturation. The reversible unfolding is detectable by multi-frequency temperature-modulated DSC.     

Ligand-induced biphasic thermal denaturation of RNAase A

DSC measurements have been accomplished in aqueous solutions of bovine pancreatic ribonuclease A (RNAase A) in the presence of subsaturating amounts of 3′ cytidine monophosphate (3′ CMP) and 2′ cytidine monophosphate (2′ CMP) atpH 5.0 and 5.5. In these conditions the experimental profiles do not conform to a one-step unfolding process. It can be emphasized, as a general phenomenon, that a strong linkage between the temperature-induced protein unfolding and the ligand binding, when the ligand is less than the saturation level, causes marked distortions from a two-state transition. A purely equilibrium thermodynamic analysis gives a correct account of this behaviour and allows to simulate calorimetric curves. It is thus possible to obtain, in an indirect manner, information about the thermodynamic parameters concerning the binding process, namely the association constant and the binding enthalpy. The values ofKb and Δ_b H for 3′CMP and 2′CMP, so determined, are consistent with the literature data.     

Equilibrium phase diagram of polystyrene and 8CB

The experimental equilibrium phase diagram of a mixture of linear polystyrene of molecular weight M_w = 44,000 g/mol and 4‐cyano‐4′‐n‐octyl‐biphenyl (8CB) is established. The three transitions smectic A‐nematic, nematic‐isotropic, and isotropic‐isotropic are observed. The first two are observed both by optical microscopy and differential scanning calorimetry (DSC) while the isotropic‐isotropic transition could be seen only via optical microscopy. Two series of samples with the same compositions were independently prepared and yielded consistent results both by microscopy and DSC. Measurements of sample compositions with less than 50 weight % of 8CB were influenced by the vicinity of the glass transition temperature (T_g) of the polymer in the mixture. This quantity is also determined by DSC as a function of composition. A single T_g is observed, which decreases with composition of the LC. Other thermodynamic quantities such as the enthalpy variations of LC in the nematic‐isotropic transition and the fraction of LC contained in the droplets are also considered. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 1841–1848, 1999     

An Aqueous Thermodynamic Model for the Complexation of Sodium and Strontium with Organic Chelates Valid to High Ionic Strength. I. Ethylenedinitrilotetraacetic Acid (EDTA)

An aqueous thermodynamic model is developed, which accurately describes the effects of Na⁺ complexation, ionic strength, carbonate concentration, and temperature on the complexation of Sr²⁺ by ethylenedinitrilotetraacetic acid (EDTA) under basic conditions. The model is developed from the analysis of literature data on apparent equilibrium constants, enthalpies, and heat capacities, as well as on an extensive set of solubility data on SrCO₃(c) in the presence of EDTA obtained as part of this study. The solubility data for SrCO₃(c) were obtained in solutions ranging in Na₂CO₃ concentration from 0.01 to 1.8 m, in NaNO₃ concentration from 0 to 5 m, and at temperatures extending to 75C. The final aqueous thermodynamic model is based upon the equations of Pitzer and requires the inclusion of a NaEDTA^3– species. An accurate model for the ionic strength dependence of the ion-interaction coefficients for the SrEDTA^2– and NaEDTA^3–aqueous species allows the extrapolation of standard state equilibrium constants for these species, which are significantly different from the 0.1 m reference state values available in the literature. The final model is tested by application to chemical systems containing competing metal ions (i.e., Ca²⁺) to further verify the proposed model and indicate the applicability of the model parameters to chemical systems containing other divalent metal-EDTA complexes.     

Thermal transitions of actin

Actin is one of the main components in the eukaryote cells which plays significant role in many cellular processes, like force-generation, maintenance of the shape of cells, cell-division cycle and transport processes. In this study the thermal transitions of monomer and polymerized actins were studied to get information about the changes induced by polymerization and binding of myosin to actin using DSC and EPR techniques. The main thermal transition of F-actin was at 67.5°C by EPR using spin-labeled actin (the relative viscosity change was around 62°C), while the DSC denaturation T _ms were at 60.3d°C for G-actin and at 70.5°C for F-actin. Applying the Lumry-Eyring model to obtain the parameters of the kinetic process and calculate the activation energy, a ‘break’ was found for F-actin in the function of first-order kinetic constant vs. 1/T. This indicates that an altered interdomain interaction is present in F-actin. The addition of myosin or heavy meromyosin (HMM) in different molar ratio of myosin to actin has changed significantly the EPR spectrum of spin-labeled F-actin, indicating the presence of the supramolecular complex. Analyzing the DSC traces of the actomyosin complex it was possible to identify the different structural domains of myosin and actin.     

Extrapolation of short-chain oligomers melting temperatures at infinite molecular weight

The determination of the thermodynamic equilibrium melting point of a polymer (T) by the extrapolation of the melting temperature of its oligomers has been extensively studied in the case of n-alkanes. Nevertheless, a recent publication¹ underlines the difficulty to realize this extrapolation. A new method is presented here, leading to an acceptable extrapolation of PE. The equation proposed may give a better value of T_m because the premelting phenomena is being considered in its development. Moreover, this method can be easily extended to a larger number of polymers, such as PEO, PEEK, PPS, etc.© 1998 John Wiley & Sons, Inc. J. Polym. Sci. B Polym. Phys. 36: 2563–2571, 1998     

The Role of Bound Water on the Energetics of DNA Duplex Melting

A combination of common and low-temperature differential scanning calorimetry (DSC) techniques was used to detect the thermodynamic parameters of heat denaturation and of ice-water phase transitions for native and denaturated DNA, at different low water contents. We suggest that the main contribution to the enthalpy of the process of the heat denaturation of DNA duplex (35±5 kJ/mol bp) is the enthalpy of disruption of the ordered water structure in the hydration shell of the double helix (26±1 kJ/mol bp). It is possible that this part of the energy composes the non-specific general contribution (70%) of the enthalpy of transition of all type of duplexes. For DNA in the condensed state the ratioα=ΔC _p/ΔS ~2 is smaller than for DNA in diluted aqueous solutions (α≅2–4). This means that there are other sources for the large heat capacity change in diluted solutions of DNA – for example the hydrophobic effects and unstacking(unfolding) of single polynucleotide chains. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molar heat capacity and thermodynamic properties of crystalline [Nd(Glu)(H<Subscript>2</Subscript>O)<Subscript>5</Subscript>(Im)<Subscript>3</Subscript>](ClO<Subscript>4</Subscript>)<Subscript>6</Subscript>·2H<Subscript>2</Subscript>O

A complex of neodymium perchloric acid coordinated with L-glutamic acid and imidazole, [Nd(Glu)(H₂O)₅(Im)₃](ClO₄)₆·2H₂O was synthesized and characterized by IR and elements analysis for the first time. The thermodynamic properties of the complex were studied with an automatic adiabatic calorimeter and differential scanning calorimetry (DSC). Glass transition and phase transition were discovered at 221.83 and 245.45 K, respectively. The glass transition was interpreted as a freezing-in phenomenon of the reorientational motion of ClO₄⁻ ions and the phase transition was attributed to the orientational order/disorder process of ClO₄⁻ ions. The heat capacities of the complex were measured with the automatic adiabatic calorimeter and the thermodynamic functions [H _T-H _298.15] and [S _T-S _298.15] were derived in the temperature range from 80 to 390 K with temperature interval of 5 K. Thermal decomposition behavior of the complex in nitrogen atmosphere was studied by thermogravimetric (TG) analysis and differential scanning calorimetry (DSC).     

Thermal properties of hafnium(IV) and zirconium(IV) β-diketonates

Five volatile hafnium(IV) and zirconium(IV) β-diketonates: hafnium(IV) acetylacetonate, hafnium(IV) trifluoroacetylacetonate, hafnium(IV) pivaloyltrifluoroacetonate, hafnium(IV) 2,2,6,6-tetramethylheptane-3,5-dionate and zirconium(IV) 2,2,6,6-tetramethylheptane-3,5-dionate were obtained, purified and identified. Thermal behavior of solid compounds was investigated by thermogravimetry (TG) and differential scanning calorimetry (DSC) in helium atmosphere and in vacuum. DSC method was also used for definition of thermodynamic characteristics of melting processes. Using the static method with quartz membrane zero-manometer and the flow method the temperature dependencies of saturated vapor pressure for hafnium(IV) complexes was obtained. The standard thermodynamic characteristics ΔH _T⁰ and ΔS _T⁰ of sublimation and evaporation processes were calculated from the temperature dependences of saturated vapor pressure.     

The thermodynamic properties of polytetrafluoroethylene

Polytetrafluoroethylenes of different crystallinity were analyzed between 220 and 700 K by differential scanning calorimetry. A new computer coupling of the standard DSC is described. The measured heat capacity data were combined with all literature data into a recommended set of thermodynamic properties for the crystalline polymer and a preliminary set for the amorphous polymer (heat capacity, enthalpy, entropy, and Gibbs energy; range 0–700 K). The crystal heat capacities have been linked to the vibrational spectrum with a θ₃ of 54 K, and θ₁ of 250 K, and a full set of group vibrations. C_v to C_p conversion was possible with a Nernst–Lindemann constant of A = 1.6 × 10^?3 mol K/J. The glass transition was identified as a broad transition between 160 and 240 K with a ΔC_p of 9.4 J/K mol. The room-temperature transitions at 292 and 303 K have a combined heat of transition of 850 J/mol and an entropy of transition of 2.90 J/K mol. The equilibrium melting temperature is 605 K with transition enthalpy and entropy of 4.10 kj/mol and 6.78 J/K mol, respectively. The high-temperature crystal from is shown to be a condis crystal (conformationally disordered), and for the samples discussed, the crystallinity model holds.