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At least 469 records · Page 26Linked to original sources
PubMed2003

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology.

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.

Animals↗
PubMed1997

Differential scanning calorimetry and small angle x-ray diffraction study of the interaction of (R)-PAF, (R)-ET-18-OME and (R)-Lyso-PAF with model membranes.

(R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF are ether phospholipid analogs exhibiting different biological activities. All three molecules are highly lipophilic and interact with cell membranes. The manner in which their interactions occur is relevant to their biological activities and may provide information on their mechanism(s) of action at the molecular level. We have studied the interactions of (R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF with model membranes using differential scanning calorimetry (DSC). A concentration-dependence study with DSC shows that all three analogs induce similar changes in the thermotropic behavior of model membranes. All produce a slight decrease in the main phase transition temperature of DPPC bilayers (deltaTc = 0.3 to 1.4 degrees C) but have a more substantial effect on the pretransition (deltaTc = 0.8 to 4.5 degrees C). All three disorder phosphatidylcholine (PC) membranes but impart order to PC/cholesterol membranes. We have used small angle x-ray diffraction experiments to supplement the DSC evidence on the topography of the ether lipid analogs in the bilayers. All three appear to insert themselves in the bilayer with their long chain parallel with the membrane bilayer chains.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Calorimetry: a tool for assessing microbial activity under aerobic and anoxic conditions.

For many years, calorimetric measurements have been used for understanding, modelling, controlling, and optimising chemical reactions. Calorimetry could be as well utilised to investigate biological processes, which however, involve very small amount of heat and therefore require very sensitive instruments. For this purpose, a Mettler Toledo RCI (Reaction calorimeter) was modified, changing both hardware and software, increasing its resolution up to 5 10m W/l. Such sensitivity allows the monitoring of aerobic and anoxic processes. This paper points out the excellent agreement between calorimetric and respirometric data, obtained simultaneously under aerobic conditions using activated sludge from a lab-scale scale reactor. Heat production rate can be directly converted in oxygen uptake rate by means of a correlation factor, whose value is approximately the same for all aerobic respiratory metabolisms. Taking into account this factor, calorimetric data were introduced in a chemical oxygen demand based model and processed for the estimation of kinetic parameters of heterotrophic biomass. Aerobic heterotrophic, denitrifying, and autotrophic nitrifying activity were determined by specific calorimetric tests. The effect of potentially toxic or inhibitory substances on the activity of all microbial communities was as well pointed out in these measurements.

Bacteria, Aerobic↗
PubMed2006

Characterization of specific donor binding to alpha1,4-N-acetylhexosaminyltransferase EXTL2 using isothermal titration calorimetry.

Glycosyltransferases encompass one of the largest families of enzymes found in nature. Their principle function is to catalyze the transfer of activated donor-sugar molecules to various acceptor substrates. The molecular basis that governs this specific transfer reaction, such as how a given transferase determines donor-sugar specificity, remains to be elucidated. Human alpha1,4-N-acetylhexosaminyltransferase (EXTL2) transfers N-acetylglucosamine and N-acetylgalactosamine but does not transfer glucose or galactose. Isothermal titration calorimetry (ITC) is a powerful technique used to characterize various binding reactions, including both protein-ligand and protein-protein interactions. ITC provides the binding stoichiometry, affinity, and the thermodynamic parameters free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) of these binding interactions. This chapter describes our ITC study demonstrating the two-step mechanism that regulates the specific binding of N-acetylhexosamines to EXTL2.

Acetylglucosamine↗
PubMed1998

Tight ligand binding affinities determined from thermodynamic linkage to temperature by titration calorimetry.

A general isothermal titration calorimetry method is described that can be used to determine equilibrium binding constants for high-affinity interactions of ligands with biological macromolecules. The method exploits the thermodynamic linkage between the ligand binding equilibrium constant and temperature. By measuring the binding enthalpy change for an interaction as a function of temperature directly, the change in affinity can be calculated with an integrated form of the van't Hoff equation that is applicable to ligand binding to biological macromolecules. When the temperature dependence of the affinity is combined with the absolute affinity determined independently at a convenient temperature (where the affinity can most accurately or most easily be measured), the absolute binding affinity over the entire temperature range is determined.

Antibodies, Monoclonal↗
PubMed1997

Polymerization of acrylic bone cement using differential scanning calorimetry.

The polymerization of acrylic bone cement using differential scanning calorimetry (DSC) was investigated. The polymerization reaction of the acrylic bone cement was found to be an approximately first order reaction. Two kinds of reaction rate constants for the polymerization reaction were observed. Both rate constants were calculated before and after the peak time. The effects of the addition of tricalcium phosphate (TCP) on the polymerization reaction of standard Surgical Simplex-P Radiopaque Bone Cement have been investigated by DSC. The TCP content had a strong retardation effect on the rate constants. The thermal stability of the acrylic bone cement was also studied by thermogravimetric analysis.

Acrylates↗
PubMed2001

Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

Arthrobacter↗
PubMed1998

Thermodynamics of ligand (substrate/end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies.

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X2<X3<X4</=X5. Contributions from the enthalpy towards the free energy change decreased with increasing chain length from X2 to X4, whereas an increase in entropy was observed, the change in enthalpy and entropy of binding being compensatory. The entropically driven binding process suggested that hydrophobic interactions as well as hydrogen bonds play a predominant role in ligand binding.

Actinomycetales↗
PubMed1999

Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.

Adenosine Diphosphate↗
PubMed2003

Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157:H7 by differential scanning calorimetry.

Differential scanning calorimetry (DSC) was used to evaluate the relative high hydrostatic pressure (HHP) resistances of bacterial strains from Staphylococcus aureus and Escherichia coli O157:H7 in vivo. The total apparent enthalpy change and thermal stability were two DSC parameters used to compare bacterial strains of untreated control and pressure-treated bacteria. DSC thermograms indicated that ribosomal denaturation appears to be a major factor in cell death by both thermal and high pressure treatments. However, the analysis of calorimetric data for control samples as well as pressure-treated samples clearly showed that the sensitivities of bacteria to various physical stresses can be different. While S. aureus 765 had a relatively higher resistance to thermal treatment in comparison to S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be utilized in the design of processes specific to targeting certain cellular components by using different physical stresses.

Calorimetry, Differential Scanning↗
PubMed1987

Efficacy of a multiple insulin injection regimen in teenagers with insulin-dependent diabetes. Carbohydrate and lipid oxidation measured by continuous indirect calorimetry.

Our study of insulin-dependent diabetic teenagers proved that a multiple insulin injection regimen (MIR) can be an acceptable and effective method of glycemic control. Further, the artificial beta cell can be used to determine insulin requirements for MIR. And finally, continuous indirect calorimetry can be used to assess metabolic control in diabetes.

Adolescent↗
PubMed1999

Indirect calorimetry in mechanically ventilated infants and children: accuracy of total daily energy expenditure with 2 hour measurements.

OBJECTIVE: To determine how accurately total daily energy expenditure can be estimated from measurement periods of less than 24 h in mechanically ventilated infants and children. DESIGN: Prospective study to validate a method to determine energy expenditure. SETTING: Pediatric intensive care unit of an university hospital. PATIENTS: Nineteen ventilated patients (age 3 weeks - 13 years) with a FiO(2)of less than 60% and tube leakage < 10% (the difference between inspiratory and expiratory tidal volume measured by the ventilator). Separately, in five patients with varying tube leakage (2-59%) the influence of tube leakage on measured energy expenditure was determined. MEASUREMENT: Energy expenditure was measured by indirect calorimetry. Total daily energy expenditure measurements for 24 h were compared with 2 h energy expenditure measurements. The influence of body temperature on measured energy expenditure was determined. RESULTS: The mean coefficient of variation among 2 hour periods of measured energy expenditure was 6% (range 2-11%) compared with total daily energy expenditure measurements. The influence of body temperature revealed an increase of 6% of energy expenditure/ degrees C. An increased tube leakage influenced the energy expenditure measurements significantly (P<< 0. 001). CONCLUSION: In a clinical situation in patients with a tube leakage << 10% total daily energy expenditure can be estimated from a 2 h measurement period.

Adolescent↗
PubMed2001

Heat does not come in different colours: entropy-enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions.

Modern techniques in microcalorimetry allow us to measure directly the heat changes and associated thermodynamics for biomolecular processes in aqueous solution at reasonable concentrations. All these processes involve changes in solvation/hydration, and it is natural to assume that the heats for these processes should reflect, in some way, such changes in solvation. However, the interpretation of data is still somewhat ambiguous, since different non-covalent interactions may have similar thermodynamic signatures, and analysis is frustrated by large entropy-enthalpy compensation effects. Changes in heat capacity (Delta C(p)) have been related to changes in hydrophobic hydration and non-polar accessible surface areas, but more recent empirical and theoretical work has shown how this need not always be the case. Entropy-enthalpy compensation is a natural consequence of finite Delta C(p) values and, more generally, can arise as a result of quantum confinement effects, multiple weak interactions, and limited free energy windows, giving rise to thermodynamic homeostasis that may be of evolutionary and functional advantage. The new technique of pressure perturbation calorimetry (PPC) has enormous potential here as a means of probing solvation-related volumetric changes in biomolecules at modest pressures, as illustrated with preliminary data for a simple protein-inhibitor complex.

Calorimetry↗
PubMed2002

Kinetics of solid state stability of glycine derivatives as a model for peptides using differential scanning calorimetry.

Kinetics of solid state stability of seven derivatives of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) of glycine as a model for amino acids and peptide drugs were studied using differential scanning calorimetry (DSC). Each DSC curve for each derivative showed an endothermic peak followed by an exothermic one, which could be attributed to the melting and decomposition, respectively. The decomposition activation energy of each derivative was calculated using the Augis and Bennet, Kissinger equations and Mahadevan approximation. Also, the melting activation energies as well as the thermodynamic parameter (enthalpy) for the investigated derivatives were evaluated. The relative stability of the derivatives in the solid state according to the calculated values of the decomposition activation energy, frequency factors and half-life for each derivative could be determined.

Calorimetry, Differential Scanning↗
PubMed2003

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry.

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.

Binding Sites↗
PubMed1997

Thermal unfolding of the N-terminal RNA binding domain of the human U1A protein studied by differential scanning calorimetry.

Thermal unfolding of the N-terminal RNA binding domain of human U1A protein (RBD1) and several variants has been observed by differential scanning calorimetry. Unfolding of the 95 amino acid domain is reversible and cooperative between pH 2.0 and 2.8 in 40 mM glycine, with a heat capacity for the transition of 1.2 kcal mol-1 K-1, and an unfolding free energy of 4.0 kcal mol-1 at pH 2.3 and 25 degrees C. At higher pH, thermal unfolding is irreversible. In contrast, unfolding of the protein by guanidine hydrochloride denaturation at pH 2.3 and pH 7.0 is reversible, with unfolding free energies of 6.6 and 9.0 kcal mol-1, respectively. DSC experiments show that RBD variants in which the N-terminal tail is truncated, or in which a functional loop is substituted, have altered unfolding free energies but little variation in their heat capacities of transition.

Amino Acid Sequence↗
PubMed2000

Titration calorimetry of surfactant-membrane partitioning and membrane solubilization.

The interaction of surfactants with membranes has been difficult to monitor since most detergents are small organic molecules without spectroscopic markers. The development of high sensitivity isothermal titration calorimetry (ITC) has changed this situation distinctly. The insertion of a detergent into the bilayer membrane is generally accompanied by a consumption or release of heat which can be measured fast and reliably with modern titration calorimeters. It is possible to determine the full set of thermodynamic parameters, i.e., the partitioning enthalpy, the partitioning isotherm, the partition coefficient, the free energy, and the entropy of transfer. The application of ITC to the following problems is described: (i) measurement of the critical micellar concentration (CMC) of pure detergent solutions; (ii) analysis of surfactant-membrane partitioning equilibria, including asymmetric insertion; and (iii) membrane-surfactant phase diagrams. Finally, the thermodynamic parameters derived for non-ionic detergents are discussed and the affinity for micelle formation is compared with membrane incorporation.

Calorimetry↗
PubMed2000

Characterization of chloroquine-hematin mu-oxo dimer binding by isothermal titration calorimetry.

Numerous studies indicate that a key feature of chloroquine's (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ-hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K(a) values) for enthalpy-driven CQ-hematin mu-oxo dimer binding fell in the narrow range of 2.3-4.4 x 10(5) M(-1). It is therefore probable that CQ-hematin mu-oxo dimer binding affinity does not diminish at the pH range (4.8-5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ-hematin mu-oxo dimer binding decreased accompanied by increased hematin mu-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5-9.0 indicates that CQ binds to two hematin mu-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin mu-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin mu-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning.

Antimalarials↗