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PubMed1991

Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry.

Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively. After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n. Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA. Peak pr at 94 degrees C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.

Bacterial Proteins↗
PubMed1991

Thermal inactivation of Listeria monocytogenes studied by differential scanning calorimetry.

The effect of NaCl on the thermal inactivation of Listeria monocytogenes has been investigated by conventional microbiological techniques and by using differential scanning calorimetry (DSC). Addition of 1.5 M-NaCl to cells grown at lower NaCl concentrations significantly increases the tolerance of cells to mild heat stress (56-62 degrees C). DSC thermograms show five main peaks which are shifted to higher temperatures in the presence of 1.5 M-NaCl. Measurement of loss of viability in the calorimeter gave good correlation between cell death and the first major thermogram peak at two NaCl concentrations. The time course of the loss of this first peak when cells were heated and held at 60 degrees C in the calorimeter matched the loss of viability, whereas the peak attributable to DNA showed little change during this process. The use of DSC to investigate the mechanisms involved in thermal inactivation is discussed.

Calorimetry, Differential Scanning↗
PubMed1999

The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: and in vivo study using differential scanning calorimetry.

Differential scanning calorimetry of whole Escherichia coil cells allowed the detection in vivo of changes in ribosome conformation. This enabled for the first time an analysis of the effects of high hydrostatic pressures on ribosomes in living cells. A correlation was observed between loss of cell viability and decrease in ribosome-associated enthalpy in cells subjected to pressures of 50-250 MPa for 20 min. Cell death and ribosome damage were therefore closely related phenomena. In pressure-treated cells, the thermogram peak temperatures decreased, suggesting that the remaining ribosomes had adopted a less stable conformation. During subsequent incubation of the cultures at 37 degrees C, peak temperatures and enthalpies gradually increased over a period of 5 h. This change in ribosome conformation had no apparent effect on cell survival, as viability continued to decrease. The addition of 5 mM MgCl2 before pressure treatment of cells prevented the reduction in stability of surviving ribosomes but had no effect on the initial loss of enthalpy or on cell viability.

Bacterial Proteins↗
PubMed2005

Water molecules in the antibody-antigen interface of the structure of the Fab HyHEL-5-lysozyme complex at 1.7 A resolution: comparison with results from isothermal titration calorimetry.

The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315-326]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A resolution. The refined structure has yielded a detailed picture of the Fab-lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an R(work) of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed.

Antigen-Antibody Complex↗
PubMed1991

Computer-controlled differential scanning calorimetry of dental composites.

In this paper, computer-controlled differential scanning calorimetry has been used to evaluate the heat liberated and the temperature rise associated with the light cure of selected dental composites. In addition, the effect of postcure heating and increased ambient temperature of light cure are examined in terms of potential secondary cure. The results indicate significant differences between the mean heats of cure of different composites, primarily reflecting the filler fraction variations of the composites. In addition, increased cure activity appears to result from both postcure heating as well as thermal activation due to higher ambient temperature of light cure. The results are analyzed and attributed to variations in resin phase composition and resin content per unit weight of composite as well as thermally induced enhancement of cure.

Calorimetry, Differential Scanning↗
PubMed2007

Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.

Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Apoproteins↗
PubMed1981

Effect of hydration on the thermal stability of protein as measured by differential scanning calorimetry. Chymotrypsinogen A.

The thermal denaturation of chymotrypsinogen A was investigated by differential scanning calorimetry in the water-content range 0.09 -1.46 g water per g protein. At water contents above 0.82 g/g, the temperature, Td, and the enthalpy, delta Hd, of denaturation were almost independent of the water content. At lower water contents, however, both Td and delta Hd showed marked dependence on the water content. The Td increased with a decrease in the water content, whereas the delta Hd decreased with a decrease in the water content in the same region. The degree of hydration dependency of delta Hd exhibited a break at approximately 0.41 g/g, which indicated that at least two types of hydration phase contributed to the thermal stability of the protein. This behavior was similar to that noted for lysozyme.

Animals↗
PubMed1982

Effect of dimethylsulfoxide and its homologues on the thermal denaturation of lysozyme as measured by differential scanning calorimetry.

The thermal denaturation of lysozyme in aqueous sulfoxide solutions (pH 3) was investigated by differential scanning calorimetry (DSC). The sulfoxides employed were dimethylsulfoxide (DMSO), ethylmethylsulfoxide, (DMSO), dimethylsulfoxide (DMSO), and butylmethylsulfoxide (BMSO). The temperature of denaturation, Td, decreased with an increase in the concentration of sulfoxide, the decrease becoming much more pronounced at higher sulfoxide concentrations. The lowering of Td was enhanced by an increase in the hydrocarbon content of the sulfoxide molecule. The enthalpy of denaturation, delta Hd, showed a complex dependence on the solvent composition; the delta Hd first increased with increasing sulfoxide concentration and then started decreasing at different concentrations for each sulfoxide. This behavior is analogous to that with monohydric alcohols, but is different from that with guanidine hydrochloride. These results may be interpreted in terms of the interactions of the sulfoxide added with the protein and with water.

Calorimetry, Differential Scanning↗
PubMed1995

Simultaneous differential scanning calorimetry, X-ray diffraction and FTIR spectrometry in studies of ovalbumin denaturation.

The new application of differential scanning calorimetry (DSC) and the susceptibility of ovalbumin to alpha-chymotrypsin gave a quantitative estimation of protein denaturation in solid ovalbumin. Solid ovalbumin in granules with 11% of water was heated at 100 degrees C in closed and nonclosed ampules. In order to compare effects of size and crystal structure, two proteins (bovine albumin and gamma-globulin) were examined at similar conditions for the extent of denaturation. Ovalbumin and bovine albumin showed similar extents of denaturation, but gamma-globulin, with a very different molecular mass, showed the maximal conformational changes. The enthalpy of denaturation was measured to elucidate the conformational changes in solid proteins. Its value was used for calculation of the degree of denaturation. The thermodynamic data associated with transition were calculated and the number of bonds broken during denaturation was determined. Intrinsic fluorescence was utilized in order to compare these two methods. Moreover, X-ray diffraction and FTIR spectrometry were applied to native and denatured proteins.

Calorimetry, Differential Scanning↗
PubMed1991

Metabolic gas exchange during aortocoronary bypass surgery using a double pump system and mechanical ventilation. A comparison between indirect calorimetry and invasive blood gas measurements using Fick's principle.

Oxygen uptake and carbon dioxide excretion during aorto-coronary bypass surgery were studied in seven patients by indirect calorimetry and compared to blood-gas based measurements. Medium-high dose fentanyl, droperidol and midazolam were used for maintaining anaesthesia. During the period of extracorporeal circulation no external oxygenator was used. Circulation was maintained by two pumps by-passing the left and right heart respectively and the patient's lungs were ventilated with O2/N2 using a Servo 900C ventilator. For indirect calorimetric measurements gas concentrations were analysed by Beckman instruments and gas volumes were measured by the Servo 900C ventilator. Oxygen uptake and carbon dioxide excretion decreased by 31% and 39%, respectively. For invasive measurements during extracorporeal circulation, arterial and venous blood gases and pump flow were used. Using pump flow instead of cardiac output when calculating oxygen uptake circumvented errors in thermodilution measurements. There was a good correlation (r = 0.88) between the invasive and the indirect calorimetric measurements. Further, there was a good correlation between naso-pharyngeal temperature and indirect calorimetric measurements of oxygen uptake (r = 0.87).

Blood Gas Analysis↗
PubMed1984

Thermal stability of human albumin measured by differential scanning calorimetry. I. Effects of caprylate and N-acetyltryptophanate.

The thermal stability of 5% previously unheated, undefatted human albumin monomer in 145 mM Na+, pH 7.0 was investigated by differential scanning calorimetry (DSC) as a function of added caprylate and/or N-acetyl-DL-tryptophanate. Caprylate was substantially more effective than N-acetyl-DL-tryptophanate in protecting the protein against thermal denaturation at a given level or at a saturating level of stabilizer. The tracing of the differential heat capacity versus temperature (thermogram) for this undefatted monomer that contained 1.5 mol endogenous, long-chain fatty acid (LCFA)/mol monomer exhibits two denaturation peaks (endotherms) in the absence of stabilizer. The endotherm with the lower denaturation temperature (Td) comprises 70% of the total heat of denaturation and also corresponds to irreversible denaturation and precipitation of 70% of the albumin. This endotherm is associated with more thermally labile protein species containing low levels of LCFA. The endotherm with the higher Td is associated with more stable protein species containing high levels of LCFA. Thus, the two endotherms are not related to the proposed domain structure of the protein but result from an uneven LCFA distribution that is due to preexisting heterogeneity in the albumin and/or heterogeneity that arises during the DSC experiment. Binding data do not support a preexisting uneven distribution of sufficient magnitude to explain the experimental results. A complementary explanation is that an uneven fatty acid distribution arises during the DSC experiment by migration of LCFA from the more labile species to the more stable as the former unfold; such migration would cause further stabilization of the latter.

Calorimetry, Differential Scanning↗
PubMed1984

Thermal stability of human albumin measured by differential scanning calorimetry. II. Effects of isomers of N-acetyltryptophanate and tryptophanate, pH, reheating, and dimerization.

Thermal stability of undefatted human albumin preparations at 5% concentration and neutral pH in 145 mM Na+ was investigated by differential scanning calorimetry. At 30 and 4 mM N-acetyltryptophanate, the thermogram for previously unheated albumin monomer is independent of the stereochemistry of this ligand; thus, the affinity of the protein must be the same for the L- and D-isomers in the temperature range of thermal denaturation (62-86 degrees C). 26 mM L-tryptophanate bestows a slight increase in stability on previously unheated monomer, whereas 27 mM D-tryptophanate has no effect, a result consistent with the reported weaker binding of the D-isomer. Previously unheated albumin monomer shows slightly greater thermal stability at pH 6.4 than at pH 7.4. The tracing of differential heat capacity versus temperature (thermogram) for monomer from once heated albumin, i.e. once processed and once heated normal serum albumin (NSA), is almost identical to that for previously unheated monomer. Monomer prepared from outdated, multiply reprocessed, multiply reheated NSA (old monomer) had the same corresponding denaturation temperatures as previously unheated monomer; the corresponding dimer (old dimer) is slightly less stable. Thermograms for old monomer and old dimer, like those for previously unheated monomer, comprise two denaturation peaks (endotherms). Their endotherms, however, are very broad, reflecting the great heterogeneity of the old proteins. The thermogram for old monomer remains broad even in the presence of N-acetyltryptophanate and/or caprylate. Old dimer does not dissociate when undergoing thermal denaturation.

Calorimetry, Differential Scanning↗
PubMed1980

A study of structurally related binding properties of concanavalin A using differential scanning calorimetry.

Differential scanning calorimetry has been used to study the interactions between concanavalin A and different carbohydrates. It was found that the binding of carbohydrate to concanavalin A stabilizes the structure of the protein as judged by an increase in transition temperature of the lectin. Furthermore, the degree of stabilization is shown to be a function of the association constant for each sugar moiety. Interactions between concanavalin A and the glycoprotein horseradish peroxidase were also studied. On changing the molar ratio of peroxidase to concanavalin A from 0.1 to 16, an initial dramatic increase in the transition temperature for concanavalin A was observed, but at ratios above four the degree of stabilization decreased; no stabilization of peroxidase was found in molar ratios above one. Results are also presented on the importance of Mn2+ and Ca2+ for the stabilization of the concanavalin A structure and for its ability to bind carbohydrates. In the presence of Mn2+ ions alone, the lectin could not form the necessary conformation for binding the carbohydrate. However, with Ca2+ ions alone the lectin was able to bind the carbohydrate ligands, as judged by the shift in its transition temperature.

Calcium↗
PubMed1982

The distribution of ubiquinone-10 in phospholipid bilayers. A study using differential scanning calorimetry.

The thermal behaviour of mixed aqueous dispersions of ubiquinone-10 with dipalmitoylglycerophosphocholine, dimyristoylglycerophosphocholine or egg phosphatidylcholine has been examined by differential scanning calorimetry. Ubiquinone-10 was found to undergo a series of thermal transitions in the heating process in the temperature range 290-300 K prior to a major melting endotherm at 317 K. The temperature of this endotherm was unaltered when the coenzyme was codispersed with phospholipid but enthalpy determinations showed that up to about 5% (mol/ol) and 20% (mol/mol) of ubiquinone-10 was removed from the transition in mixtures with dimyristoyl and dipalmitoyl derivatives of phosphatidylcholine respectively. Likewise the pre-transition and main endothermic transitions of the saturated phospholipids were largely unperturbed by the presence of ubiquinone-10 but up to 22% (mol/mol) and 8% (mol/mol) of the dimyristoyl and dipalmitoyl derivatives of phosphatidylcholine respectively were removed from the endotherm. These data suggest that at molar percentages less than 20% ubiquinone-10 a high proportion of the coenzyme is molecularly mixed within the phospholipid bilayer but as the proportion increases there is a greater tendency for the coenzyme to form aggregates that display typical melting and crystallization behaviour.

Calorimetry, Differential Scanning↗
PubMed1988

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry.

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.

Animals↗
PubMed1990

Differential scanning calorimetry of lobster haemocyanin.

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.

Animals↗
PubMed1990

Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier-transform infrared spectroscopy and differential scanning calorimetry.

The phase behaviour, particularly the fluidity within each phase state and the transitions between them, of lipopolysaccharides and of their lipid moiety, free lipid A, of various species of Gram-negative bacteria, especially of Salmonella minnesota and Escherichia coli, has been investigated by applying mainly Fourier-transform infrared spectroscopy and differential scanning calorimetry. For enterobacterial strains, the transition temperatures of the gel----liquid crystalline (beta----alpha) phase transition of the hydrocarbon chains in dependence on the length of the sugar moiety are highest for free lipids A (around 45 degrees C) and lowest for deep rough mutant lipopolysaccharides (around 30 degrees C). Evaluating certain infrared active vibration bands of the hydrocarbon moiety, mainly the symmetric stretching vibration of the methylene groups around 2850 cm-1, it was found that, in the gel state, the acyl chains of lipopolysaccharides and free lipid A have a higher fluidity as compared with saturated and the same fluidity as compared with unsaturated phospholipids. This 'partial fluidization' of lipopolysaccharide below the transition temperature correlates with its reduced enthalpy change at that temperature compared to phospholipids with the same chain length. The fluidity depends strongly on ambient conditions, i.e. on the Mg2+ and H+ content: higher Mg2+ concentrations and low pH values make the acyl chains of free lipid A and lipopolysaccharide preparations significantly more rigid and also partially increase the transition temperature. The influence of Mg2+ is highest for free lipid A and decreases with increasing length of the sugar side chain within the lipopolysaccharide molecules, whereas the effect of a low pH is similar for all preparations. At basic pH, a fluidization of the lipopolysaccharide and lipid A acyl chains and a decrease in transition temperature take place. Free lipid A and all investigated rough mutant lipopolysaccharides exhibit an extremely strong lyotropic behaviour in the beta----alpha melting enthalpy but not in the value of the transition temperature. The phase transition is distinctly expressed only at water concentrations higher than 50-60%. A further increase of the water content still leads to an increase in the phase-transition enthalpy, particularly for lipopolysaccharides with a more complete sugar moiety. The fluidity of the hydrocarbon chains is shown to be an important parameter with respect to the expression of biological activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗