Scanning Microcalorimetry Research Articles

High-Pressure Scanning Microcalorimetry – A New Method for Studying Conformational and Phase Transitions

The development of high-pressure scanning microcalorimetry and the first results studying transitions in proteins, lipids, and model polymers are reviewed. Special attention is given to changes (increments) in volume parameters upon transitions as well as the nature of these changes. It is demonstrated that the use of the model of compound transfer reaction in its purest form for assessment of denaturation volume effects failed due to serious difficulties.

Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

Lysozyme Stabilization under High Pressure: Differential Scanning Microcalorimetry

The heat denaturation of lysozyme has been studied by high-pressure differential scanning microcalorimetry. It has been demonstrated that an increase in pressure has different influence on denaturation temperature and enthalpy at different pH values. It has been established that the pressure increase has no appreciable effect on the transition cooperativity. The experimental data have been analyzed using an equilibrium model of transition between two states. Partial molar volume changes accompanying the denaturation as well as isothermal compressibility and thermal expansibility coefficients have been assessed. In contrast to the denaturation of most globular proteins, the lysozyme denaturation under conditions of the experiment was accompanied by positive volume changes. Possible reasons for this unusual behavior have been discussed.

Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (Nop), whereas triiodothyronine decreases it 10-fold at −50mV. Rose Bengal, phloxine B and erythrosin significantly increase Nop by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60mV at 50μM, while Rose Bengal, phloxine B and erythrosin at 2.5μM reduce the membrane dipole potential by 120, 80 and 50mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.

Orientational disorder and phase transitions in ammonium oxofluorovanadates, (NH4)3VOF5 and (NH4)3VO2F4

Single crystals of (NH4)3VOF5 and (NH4)3VO2F4 were obtained from aqueous fluoride solutions and phase transitions in these compounds were investigated using X-ray diffraction, differential scanning microcalorimetry (DSM) and vibrational spectroscopy. The room-temperature (RT) phases of these compounds belong to orthorhombic symmetry [Immm and I222, Z = 6, for (NH4)3VOF5 and (NH4)3VO2F4, respectively] with similar unit-cell parameters and two independent vanadium atoms. Above RT [at 350 and 440 K for (NH4)3VOF5 and (NH4)3VO2F4, respectively], the compounds undergo reversible phase transitions into high-symmetry dynamically disordered elpasolite-like (Fm{\bar 3}m, Z = 4) structures with six and 12 spatial orientations of the vanadium octahedron for (NH4)3VOF5 and (NH4)3VO2F4, respectively. The ligand atoms are distributed in a mixed (split) position of 24e + 96j, one of the ammonium groups is disordered on the tetrahedron 32f site, but another one forms eight spatial orientations due to disorder of its hydrogen atoms in the 96j position. DSM and spectroscopic data enable the phase transition from high temperature to room temperature to be connected with the transition from isotropic orientations of the octahedron to its two different dynamic states.

The effects of allantoin, arginine and NaCl on thermal melting and aggregation of ribonuclease, bovine serum albumin and lysozyme

Allantoin is widely used as a skin care agent and readily forms crystals, which were recently shown to bind endotoxins and high molecular weight aggregates in cell culture harvests. Here, we have investigated the effects of allantoin on thermal stability and aggregation of protein using ribonuclease, bovine serum albumin and lysozyme using temperature-regulated circular dichroism (CD) and differential scanning microcalorimetry (DSC).Ribonuclease showed no change in thermal stability and aggregation by the addition of allantoin. While allantoin showed no effects on the thermal stability of bovine serum albumin, it enhanced aggregation. Similarly, allantoin showed no stabilizing effects on lysozyme, but it strongly suppressed aggregation. Such suppressed aggregation resulted in reversibility of thermal unfolding of lysozyme. These effects of allantoin were then compared with those of NaCl and arginine hydrochloride. Arginine was similar to allantoin at low concentrations, where both solvent additives can be compared due to limited solubility of allantoin.

Studying of drug solubility in water and alcohols using drug-ammonium ionic liquid-compounds

Synthesis of three mefenamic acid (MEF) derivatives - ionic liquid compounds composed of MEF in an anionic form and ammonium cation (choline, MEF1), or {di(2-hydroxyethyl)dimethyl ammonium (MEF2)}, or {tri(2-hydroxyethyl)methyl ammonium compound (MEF3)} is presented. The basic thermal properties of pure compounds i.e. fusion temperatures, and the enthalpy of fusion of these compounds have been measured with differential scanning microcalorimetry technique (DSC). Molar volumes have been calculated with the Barton group contribution method. The solubilities of MEF1, MEF2 and MEF3 using the dynamic method were measured at constant pH in a range of temperature from (290 to 370) K in three solvents: water, ethanol and 1-octanol. The experimental solubility data have been correlated by means of three commonly known GE equations: the Wilson, NRTL and UNIQUAC with the assumption that the systems studied here present simple eutectic behaviour. The activity coefficients of pharmaceuticals at saturated solutions in each binary mixture were calculated from the experimental data. The formation of MEF-ionic liquid compounds greatly increases the solubility in water in comparison with pure MEF or complexes with 2-hydroxypropyl-β-cyclodextrin. The development of these compounds formulations will assist in medication taking into account oral solid or gel medicines.

Macromolecular complexes of lysozyme with kappa carrageenan

We present a structural study of the complexation and binding of lysozyme (Lys) with kappa carrageenan (kCG) by means of turbidity measurements, phase analysis, dynamic and electrophoretic light scattering, differential scanning microcalorimetry (DSMC), confocal laser scanning (CLSM) microscopy, fluorescence and circular dichroism measurements. Complexation is governed by both electrostatic interactions and secondary forces, and exhibits a maximum at the kCG to Lys ratio for which mutual compensation of charges occurs. The effect of the ionic strength (I) on complexation has a nonmonotonous character displaying a maximum in complex formation at I ≈ 0.03. The specific pH value at which complex formation is completely suppressed (pHSet), is only slightly dependent on the I value. Turbidity measurements indicate complexation of Lys with kCG at a pH as high as 11.5 (I = 0.01). Molecules of Lys are placed mainly on the periphery of the complex particles and the localization of kCG has an irregular character without formation of a single center of binding. Complexation in dilute solutions leads to a spectacular increase in the helix content, whereas in semidilute solutions complexation causes a decrease of the temperature of denaturation, suggesting that kCG has a higher affinity for the unfolded state than for the native state of Lys.

Study on the physicochemical properties of quinoa starch

Chenopodium quinoa Willd., as a pseudocereal, has a variety of nutritional components, and has medicinal and hygienic value.In this work, X-ray diffraction, scanning electron microscopy ( SEM) and differential scanning microcalorimetry ( DSC) were utilized to analyze the structure and physicochemical properties of quinoa starch, which was prepared by alkaline method and enzyme method, respectively. Meanwhile, the freezing thaw stabilities, swelling power and iodine blue values of quinoa starch were detected.The results showed that granules of quinoa starch were small, with diameter ranging from 1.15 to 1.97 μm, and had more branched-chain starch. The swelling power, solubility, freeze-thaw stability, anti-aging properties, translucency and gelatinization temperature of these starches were significantly correlated with their respective structural features.

The structure and phase transitions in oxyfluoride (ND4)2MoO2F4

Single crystals of (ND4)2MoO2F4 containing 95% deuterium were produced by repeated recrystallization of (NH4)2MoO2F4 in heavy water. The effect of deuteration on the parameters of successive phase transitions was investigated by differential scanning microcalorimetry and polarization optics. X-ray analysis of the original and distorted phases was performed. It was found that deuteration does not change the sequence of crystal phases typical for (NH4)2MoO2F4, but leads to a shift in the phase transition Cmcm → Pnma towards the tricritical point. The mechanism of phase transitions in (ND4)2MoO2F4 was associated with both the ordering and the displacement of atoms.

Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling.

The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements, and differential scanning microcalorimetry (μDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational changes to albumin if it is organized in a micellar form, while albumin influences the self-assembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The μDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the macromolecules, with the emerging results projecting potential applications linked to reaching optimal conditions for certain drug formulations.

Physicochemical and thermodynamic properties of the {1-alkyl-1-methylmorpholinium bromide, [C1Cn=3,4,5MOR]Br, or 1-methyl-1-pentylpiperidinium bromide, [C1C5PIP]Br + water} binary systems

In this study, the density, ρ, dynamic viscosity, η, (solid+liquid), SLE and (vapor+liquid), VLE phase equilibria of four binary systems (ionic liquid, IL+water) are determined. Heat capacities, Cp of (organic salt+water) mixtures were studied as well. In this work, a series of organic salts namely: 1-mehyl-1-propylmorpholinium bromide, [C1C3MOR]Br, 1-mehyl-1-butylmorpholinium bromide, [C1C4MOR]Br, 1-mehyl-1-pentylmorpholinium bromide, [C1C5MOR]Br and 1-methyl-1-pentylpiperidinium bromide, [C1C5PIP]Br have been synthesized. The structure of the synthesized compounds were confirmed by 1H NMR, 13C NMR and elemental analysis. The thermophysical characterization of pure organic salts, including: temperature of phase transition (Ttr), enthalpy of phase transition (ΔtrH), melting temperature (Tm) and enthalpy of melting (ΔmH) have been determined using differential scanning microcalorimetry technique (DSC) at atmospheric pressure. Additionally, the thermogravimetry technique (TG/DTA) was used to determine the decomposition temperature. The influence of the alkyl chain length in the morpholinium cation as well as the cation structure on the physicochemical and thermodynamic properties presented in this work are discussed.

Proteins in binary solvents.

Proteins in living organisms exist in complex aqueous solutions or embedded in membranes. In solution, proteins are surrounded by a tightly bound hydration layer, which is more ordered and less mobile than bulk water. As a consequence, water plays a major role in controlling protein structure stability, conformational flexibility, dynamics, and functionality, but it also appears that protein surface regulates the structuring of the surrounding water. The presence of cosolvents can modify the hydration layer characteristics and then the whole protein structural and dynamical properties. Because cytoplasm or biological liquids are complex solutions, the knowledge of the solvation shell characteristics in mixed solvents should be considered as a crucial step in describing biological processes at molecular level. This review reports on recent studies on the structural and thermodynamic properties of model proteins dissolved in binary solvent mixtures by small-angle neutron scattering (SANS) and differential scanning microcalorimetry (DSC) techniques. We will show that contrast variation SANS experiments allow to acquire a direct knowledge of both protein structure and protein solvation shell (in terms of low-resolution shape and solvent/cosolvent composition), while DSC experiments provide information on all the relevant thermodynamic properties. We will focus on two main points. First, an extended description of the thermodynamic model used to define the equilibria between water and cosolvent molecules in the protein solvation shell will be presented. Second, the determination of the peculiar characteristics of the protein solvation layer, which will be illustrated by considering different systems. As a conclusion, we will show that the investigation of structure and thermodynamics of proteins in binary aqueous mixtures is an important way to understand the role of hydration in protein stability and activity.

Static and dynamic behaviour of responsive graphene oxide-poly(N-isopropyl acrylamide) composite gels.

Thermoresponsive hydrogels have enormous potential e.g., as sensors, actuators, and pollution control remedies or in drug delivery systems. Nevertheless, their application is often restricted by physical limitations (poor mechanical strength and uncontrolled thermal response). Composite systems may offer a means of overcoming these limitations. This paper presents a systematic study of the structure and dynamics of graphene oxide-poly-(N-isopropylacrylamide) composite systems, and investigates the effect of the nanoparticle filler content on the mechanical and swelling properties of the systems. A combination of macroscopic (swelling and elastic modulus) and microscopic (differential scanning microcalorimetry, small angle neutron scattering and neutron spin-echo spectroscopy) investigations reveals that the architecture of the polymer network is modified by chain nucleation at the surface of the GO platelets, and these form a percolating network inside the gel. Our results show that the elastic modulus of the gels is reinforced by the filler, but the mobility of the polymer chains in the swollen state is practically unaffected. The macroscopic deswelling of the composites, however, is slowed by the kinetics of ordering in the GO network.

Study of phase equilibria and the physicochemical properties of selected pharmaceuticals

In this work the phase equlibria in binary system: (Drug+solvent) was examined and their physicochemical properties were analyzed. The investigated substances were: synephrine (SYN), isoprenaline hydrochloride (IPR) and metaraminol bitartrate (MET). Solubility of this drugs was determined in three solvents: water, ethanol and 1-octanol. The molecular structure of these drugs is based on a phenthylamine skeleton, therefore all studied pharmaceuticals have an aromatic structure with different amounts of hydroxyl groups located at an aromatic ring. They also have different substituents on the amino group inside the chemical chain. The amino-group confers basic properties of the molecule, whereas the phenolic OH group is weakly acidic. The tested substances belong to the group of adrenergic drugs (adrenomimetics) and stimulate the sympathetic nervous system.Solubilities of three drugs were measured in the temperature range from 270K to 450K at constant pH. All of studied systems were described as simple eutectic phase diagrams, with complete miscibility at liquid phase and non-miscibility in solid phase. The results are presenting as two phases in equilibrium as a function of temperature. These studies are necessity when the drugs are introducing into the pharmaceutical field. The obtained experimental data were correlated with three equations: Wilson, NRTL and UNIQUAC. Moreover dissociation constants and corresponding pKa values of drugs were obtained with Bates–Schwarzenbach method at temperature 298.15K in the buffer solutions.The DSC (differential scanning microcalorimetry) was used to measure thermal basic properties of pure drugs, that is: the glass-transition temperature, temperature of melting, enthalpy of fusion and the heat capacity at the glass-transition temperature.

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages.

We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages λ and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals. A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage λ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.

Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor.

We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75Å (complex with squalene) and 1.6Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.

Moderate pressure has no distinct impact on hydrophobic hydration of proteins

The effect of high pressure on the change in the heat capacity (Δcp) of ribonuclease A and lysozyme upon denaturation has been studied using scanning microcalorimetry techniques. It has been shown that the pressure has no impact on Δcp up to 178MPa, although appreciably varies the denaturation enthalpy. This result confirms the thermodynamic similarity of the denaturation process and the hydration degree of polypeptide chain in the denatured state at ambient and elevated pressure. Our findings are in conflict with some hypotheses concerning the high pressure effect on the structure stability of proteins.

Interactions in aromatic probe molecule loaded poly(N-isopropylacrylamide) hydrogels and implications for drug delivery

Small aromatic molecules are known to interact with poly(N-isopropylacrylamide) (PNIPA) based hydrogels, one of the most frequently employed polymers in temperature induced drug delivery systems. These interactions are poorly understood at the molecular level. In this article we investigate PNIPA both at the macroscopic and at the molecular level using measurements of swelling, differential scanning microcalorimetry (DSC), X-ray powder diffraction (XRD) and solid state 1H NMR methods. The nature and the strength of the interactions affect the efficiency and kinetics of drug delivery. Phenols exert a major influence on PNIPA by reducing its phase transition temperature. The effect depends linearly on the phenol concentration, and is influenced also by the number of phenolic OH groups, as well as their relative positions. The strong interaction between phenol and the polymer that is detected by NMR hinders the crystallisation of phenol when the water is gradually evaporated. The aminoethyl phenol derivative dopamine has a much more limited effect, but in the opposite direction – the transition temperature increases slightly. The strong interaction observed among the dopamine molecules disables the polymer–dopamine interaction and favours crystallization of the dopamine when water is removed. These results reveal that embedding the drugs into polymer matrices for controlled delivery can alter the crystallinity of the stored molecules. As morphology is one of the crucial factors in delivery, this may compromise the rate and the efficiency of release.

Solubility of pharmaceuticals in water and alcohols

The aim of this study was to examine the phase equilibria in binary systems: {pharmaceutical, Ph (1)+water, or alcohol (2)}. This work was carried out for four pharmaceuticals, Phs: aminophylline and (−) lobeline hydrochloride, perphenazine and indomethacin. Solubility of tested Phs was determined in three solvents. Water, the main component of any living organism. Ethanol, substance corresponding to the transport of the Ph in the body, and 1-octanol, a model compound of human cell and skin membrane. All studied Phs have aromatic structure and functional groups typical for drugs.The differential scanning microcalorimetry technique, DSC was used to measure basic thermal properties of pure drugs, i.e., temperatures of fusion, enthalpy of fusion, glass-transition temperatures and heat capacity change at the glass-transition temperature. Molar volumes have been calculated with Barton group contribution method. The Bates–Schwarzenbach method enabled us to determinate the pKa of used Phs. These values are important because it may designate the Phs dosage and the activity at the certain pH. The pKa experimental values are slightly different than the literature data which was already published as a results of different experimental methods. In this work, altogether 12 binary systems {Ph (1)+solvent (2)} were studied with the use of dynamic and spectrophotometric method. Three GEx thermodynamic models were used to correlate the experimental data: Wilson equation, NRTL equation and UNIQUAC equation.