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Thermal transition studies of a mature xanthelasma by differential scanning calorimetry.

A large xanthelasma which had been present for at least 5 years was removed surgically from a normolipaemic female age 54 years, and examined in the fresh state by differential scanning calorimetry. Thermal transitions recorded over the range 30-40 degrees C suggest that the lipid present, predominantly esterified cholesterol, is not bound to protein or other tissue components, and that the chronicity of mature xanthelasmata as with ectopic lipid deposits at other sites is enhanced by chemical modification of lipid and effects on component phase behaviour, which are significant at local skin temperature.

Calorimetry, Differential Scanning↗

Reversible thermal transition of brain myelin proteolipid. A preliminary report on a high-sensitivity differential scanning calorimetry study.

Brain myelin proteolipid has been investigated using high-sensitivity differential scanning calorimetry (DSC) under various conditions. Crude proteolipid with a 40% (w/w) content of protein gave rise to a reversible transition, centered at about 60 degrees C. The specific enthalpy of the transition was 50 +/- 5 J X g-1 with a calorimetric to van't Hoff enthalpy ratio of 5.7 +/- 0.5. To our knowledge this is the first intrinsic membrane protein in which a reversible thermal transition has been detected and investigated by DSC. Similar experiments were carried out using the recombinants of delipidated proteolipid and the pool of natural membrane lipids; in this case the transition was less enthalpic and showed lower cooperativity. The recombinants with lecithins, however, did not show any transition at 60 degrees C.

Animals↗

Comparative study of isomalt and sucrose by means of continuous indirect calorimetry.

Isomalt (Palatinit) an equimolar mixture of alpha-D-glucopyranosido-1,6-sorbitol and alpha-D-glucopyranosido-1,6-mannitol, was compared to sucrose in a prospective double-blind controlled crossover study. The acute effects of oral ingestion of 30-g loads of isomalt or sucrose on plasma glucose, insulin, free fatty acids (FFA), lactic acid, and carbohydrate (CHO) and lipid oxidation were studied over six hours by means of continuous indirect calorimetry in ten healthy normal-weight subjects. Unlike sucrose, whose ingestion was followed by significant changes in plasma glucose, insulin, and lactic acid during the first 60 minutes of the test, no significant changes in these parameters were observed following the administration of isomalt. The increase in CHO oxidation occurring between 30 and 150 minutes was significantly lower (P less than 0.01) following isomalt than after sucrose. Conversely, the decrease in lipid oxidation was significantly less (P less than 0.01) after isomalt in comparison to sucrose. It is concluded that the rise in CHO oxidation and in plasma glucose and insulin levels is markedly reduced when sucrose is replaced by an equal weight of isomalt. In contrast to other sugar substitutes, no increase in plasma lactic acid was observed after isomalt administration.

Adult↗

Substrate oxidation errors during combined indirect calorimetry-hyperinsulinemic glucose clamp studies.

The procedure of indirect calorimetry is often combined with the hyperinsulinemic, euglycemic clamp technique so that intracellular rates of glucose oxidation (Gox), fat oxidation (Fox), and energy expenditure (EE) can be determined at different insulin concentrations and rates of whole-body glucose uptake. In order to perform these calculations, rates of protein oxidation (Pox) must be known and are usually estimated from urinary nitrogen (N) excretion. The use of urinary N assumes that this measurement accurately reflects Pox and is unaltered by the glucose clamp technique. To examine these assumptions and determine potential errors in rates of Gox, Fox, and EE with this method, eight healthy subjects each had basal urinary N excretion determined on 4 different days and during a 300 pmol/m2/min hyperinsulinemic, euglycemic clamp. Mean basal urinary N excretion was 6.4 +/- 1.6 mg/min. Within individuals, basal urinary N was highly variable on the 4 different days with a mean coefficient of variation (CV) of 36% +/- 18%. Over the range of basal respiratory quotient (RQ) values in this study (0.78 to 0.85) the day-to-day variation in basal urinary N resulted in potential errors of 11% to 23% for Gox, 16% to 24% for Fox, but minimal effects (less than or equal to 1%) on EE. During the hyperinsulinemic, euglycemic clamp, RQ increased to 0.95 or greater, while urinary N excretion, rather than decreasing as expected, increased by 47% (6.4 +/- 1.6 to 9.4 +/- 2.8 mg/min) due in part to increases in urea clearance from 37.5 +/- 6.7 to 75.2 +/- 12.4 mL/min (P less than .025). This increased urinary N excretion had minimal influence on Fox and EE, but underestimated Gox by up to 5% at RQ less than 0.95. A more accurate estimate of urinary N excretion during hyperinsulinemic clamps may be obtained by correcting for changes in urea clearance. These results indicate that basal urinary N excretion is highly variable and influenced by hyperinsulinemic glucose clamps. Thus, urinary N excretion, particularly during the basal state, may not accurately reflect changes in Pox and can lead to substantial errors in Gox and Fox.

Adult↗

Turnip yellow mosaic virus and its capsid have thermal stabilities with opposite pH dependence: studies by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy.

In the differential scanning calorimetry (DSC) scans of turnip yellow mosaic virus (TYMV) or its capsid a single endotherm was observed. The endotherm was attributed to disruption of the virion or capsid structure with accompanying protein denaturation. At pH 4.5 the thermal stabilities of the TYMV virion and capsid were similar. With increasing pH, the capsid stability increased while the virion stability decreased. At neutral pH the capsid disrupted at 83.5 degrees, and the virion disrupted at 69 degrees. Our results suggest that packaging of viral RNA in the TYMV capsid imparts instability. The pHmid for disruption of the TYMV capsid is 5.7, which is in the pKa range expected for histidine side chains. Hence repulsive interactions involving one or more of the three histidines of the TYMV coat protein may explain the decreased stability of the TYMV capsid at low pH. This conclusion is supported further by the observation that belladonna mottle virus (BDMV) capsid (BDMV and TYMV belong to the tymo virus group), which contains no histidine in its coat protein, did not exhibit pH-dependent stability. The size of the cooperative unit in the disruption of TYMV capsid was estimated to be approximately that of a dimer of the coat protein, at pH 7.0, but a larger oligomer at low pH. Several reports implicate pH-dependent protein-RNA interactions with a pHmid near 7 as important in stabilizing tymovirus virions. Both DSC and 31P nuclear magnetic resonance linewidth analyses of the TYMV virion showed a transition midpoint at pH 7.0.

Calorimetry, Differential Scanning↗

Thermal stability of cowpea mosaic virus components: differential scanning calorimetry studies.

Differential scanning calorimetry studies show that the thermal stability of cowpea mosaic virus (CPMV) is pH dependent in the pH range 5 to 8. The four different components of CPMV, top (protein shell devoid of RNA), middle (virus particle containing the smaller RNA), bottom upper and bottom lower (virus particle containing the larger RNA), are stable up to 64-70 degrees at pH 5.0 and up to 43-46 degrees at pH 8.25. These components did not exhibit any difference in their thermal stability between pH 6.0 and 8.0.

Calorimetry, Differential Scanning↗

Physicochemical studies of two major subpopulations of low density lipoproteins by differential scanning calorimetry and n.m.r. spectroscopy.

Two subpopulations, layer 2 (density 1.025-1.029 g/ml) and layer 3 (density 1.032-1.043 g/ml) of low density lipoproteins (LDL) were isolated from fresh human plasma of normal lipidaemic subjects by density gradient ultracentrifugation. Chemical analyses demonstrated the ratios of triglyceride/cholesterol ester decreased with increasing densities of subfractions. These subfractions together with triglyceride-rich lipoproteins (layer 1, density less than 1.019 g/ml) were subjected to physicochemical studies by differential scanning calorimetry (d.s.c.) and nuclear magnetic resonance (n.m.r.) spectroscopy. The average transition temperature (Tt) of layer 2 was 34.20 +/- 0.83 degrees C and that of layer 3 was 37.25 +/- 0.35 degrees C. In addition, many of the layer 3, but not layer 2 and layer 1, samples showed structural alteration and gave rise to an average Tt of 39.18 +/- 1.24 degrees C. The structural alteration could be detected with polarizing light microscopy showing birefringent spherulites at body temperature. The peak Tt values obtained by d.s.c. were in good agreement with those by n.m.r. spectroscopy. These results demonstrate the physicochemical heterogeneity within the LDL density region and suggest that layer 3 subpopulation is much more labile than the others.

Adult↗

Kinetics of collagen denaturation in mammalian lens capsules studied by differential scanning calorimetry.

Calorimetry was used to measure the rates of irreversible denaturation of collagen in intact mammalian lens capsules, isothermally stored in water at different temperatures, and the kinetic characteristics of the process determined. At 47.4 degrees C, the number of native collagen molecules declined by a factor of 10 in 21.4 +/- 2.9 min (this is called the D-value) and increasing the storage temperature by 2.32 +/- 0.12 degrees C reduced the D-value by a factor of 10. The activation energy of the process was 860 +/- 44 kJ mol-1, and the activation enthalpy and entropy were respectively 858 +/- 45 kJ mol-1 and 2.38 +/- 0.14 kJ mol-1 K-1. Using equations derived in the paper, these kinetic characteristics were used to predict the position and shape of the denaturation endotherms over a range of scanning rates. Comparison of the predicted and actual positions of peak maxima showed a close correspondence, demonstrating that the principal phenomenon determining the position of the peak maxima was an irreversible rate process. While there was fairly good agreement between the predicted and actual shape of the endotherm at low scanning rates, measurements at high rates were distorted by the finite response time of the calorimeter, and the agreement was poor.

Animals↗

Conformations and stabilities of human Glu1- and Lys78-plasminogen and of the fragments mini- and microplasminogen, analysed by circular dichroism and differential scanning calorimetry.

The conformations and stabilities of two forms of human plasminogen, Glu1-plasminogen (Glu1-HPg, Glu1-Asn791) and Lys78-plasminogen (Lys78-HPg, Lys78-Asn791), and two enzymatically derived plasminogen fragments, miniplasminogen (mini-HPg, Val443-Asn791) and microplasminogen (micro-HPg, Lys531-Asn791) were analysed by circular dichroism and differential scanning calorimetry. The two plasminogen forms differ by the lack of 77 N-terminal amino acids in Lys78-HPg in comparison to Glu1-HPg. Mini-HPg is composed of kringle 5 and the protease domain of HPg whereas micro-HPg is built from the protease domain of HPg and a stretch of about 15 amino acids from kringle 5. Differential scanning calorimetric measurements of Glu1-HPg and Lys78-HPg reveal seven thermal transitions for both plasminogen forms. The results obtained for Lys78-HPg largely agree with recently published data (Novokhatny, V. V., Kudinov, S. A. and Privalov, P. L. J. Mol. Biol. 1984, 179, 215). Three thermal transitions corresponding to kringle 5 and to two subdomains of the C-terminal protease region were identified for mini-HPg. In micro-HPg, the two thermal transitions of the protease region were found but one of the protease subdomains was modified and its stability was much higher than in any of the other studied proteins. According to the microcalorimetric data obtained for mini-HPg and micro-HPg, transitions 5 and 6 of Glu1-HPg and Lys78-HPg were reassigned to kringle 5 and to a subdomain of the protease region, respectively, in contrast to literature data.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

Differential scanning calorimetry to study the possible ternary complex formation between chlorhexidine, phosphatidylcholine and some nonionic tenzides.

The effect of chlorhexidine and its molecular complexes with some nonionic tenzides (Tween and Myrj series) on the phase transfer parameters of dipalmitoylphosphatidylcholine has been studied by differential scanning calorimetry. The application of principal component analysis facilitated the evaluation of the experimental data matrix. It was found that both the lipophilicity of the tenzide and the strength of interaction between chlorhexidine and nonionic tenzide had a considerable impact on some of the phase transition parameters of dipalmitoylphosphatidylcholine. These findings indicate the possible existence of ternary complexes. This ternary complex formation may cause a decrease of chlorhexidine activity in the presence of nonionic tenzides.

Calorimetry, Differential Scanning↗

Large increase in thermal stability of the CH2 domain of rabbit IgG after acid treatment as evidenced by differential scanning calorimetry.

Rabbit IgG after exposure to 0.05 M glycine-HCl, pH 2.0, and native IgG were compared by differential scanning calorimetry (DSC) at pH 3.5 and C1q binding studies at pH 7.8. For acid-treated IgG, a large increase (by approx. 12-15 degrees C) in thermal stability of the CH2 domain occurs and this domain no longer demonstrates a separate and thermodynamically independent unfolding at 56 degrees C seen for native IgG. The results suggest that stabilization of the CH2 domain in acid-treated IgG arises from stronger, relative to the native protein, interaction of the CH2 domain with adjacent and more stable IgG domain(s). Conformational differences of the two forms of IgG were confirmed at neutral pH by a 4-fold increase of C1q-binding affinity of acid-treated IgG.

Acids↗

A differential scanning calorimetry analysis of the age-related changes in the thermal stability of rat skin collagen.

Differential scanning calorimetry (DSC) has been applied to the study of connective tissue to evaluate the denaturation process of collagen. We have applied this technique to the study of the ageing of rat skin. We have tried to correlate the variations of the parameters measured by DSC and the modifications of collagen crosslinks with ageing. The thermograms obtained are composed of one main peak located between two shoulders. The relative size of each peak varies with time: the first peak diminishes regularly from 2 to 20 months whilst, at the same time, the third peak increases; the recovery temperature increases with age (+ 16 degrees C between 2 and 20 months); the total denaturation enthalpy does not vary: the main value obtained is 5.9 X 10(-2) J/mg collagen. On the other hand, the assay of reducible collagen crosslinks in rat skin, over the same age range, shows a decrease of heat-labile aldimine crosslink (essentially hydroxylysinonorleucine). These results and the study of thermograms obtained with altered rat skin (animals treated with beta-aminopropionitrile or skin reduced with NaBH4) allow us to conclude that heat-labile and heat-stable crosslinks account for a collagen thermal stabilization which can explain the delay of denaturation characterized by the third peak of DSC thermograms.

Aging↗

Changes in structure of alpha 2-macroglobulin upon reaction with trypsin as assessed by light scattering and differential scanning calorimetry.

Employing a combination of static and dynamic light scattering, as well as differential scanning calorimetry (DSC), the structural changes which appear in alpha 2-macroglobulin (alpha 2M) upon trypsin binding have been further characterized. Light-scattering measurements suggest that a 15% reduction in both the hydrodynamic radius and radius of gyration occurs when two molecules of trypsin complex to alpha 2M. Approx. 85% of this trypsin-induced compaction results from the binding of the first proteinase. A complementary result was obtained from DSC measurements in which the major fraction of the trypsin-induced conversion of alpha 2M to a single more thermally stable form results from interaction with the first proteinase molecule. These observations support a functionally asymmetric model of trypsin binding to alpha 2M in which the significant reduction in size of the complex is primarily due to the initial interaction of alpha 2M with a single proteinase molecule.

Calorimetry, Differential Scanning↗

Fourier transform infrared spectroscopy and differential scanning calorimetry of transferrins: human serum transferrin, rabbit serum transferrin and human lactoferrin.

Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) have been used to investigate the solution structure and thermal stability of human serum transferrin (HST), human lactoferrin (HLF) and rabbit serum transferrin (RST) in their diferric and apo forms. Our study shows that: (A) The secondary structure of all the proteins studied (estimated in H2O) was in the range 43-53% alpha-helix and 23-28% beta-sheet. These values differ markedly from previously reported circular dichroism (CD) data. This is attributed to the fact that FTIR and CD measure different aspects of secondary structure (hydrogen bonding and dihedral angles, respectively). (B) The secondary structural content of the proteins is not altered by iron binding or release. However, the iron-free proteins undergo a greater extent of 1H-2H exchange than the diferric proteins indicating that significant structural changes do occur upon iron binding/release. (C) The removal of iron leads to thermal destabilization of HST, HLF and RST. Structural variation in the apo transferrins is indicated by the observation of a single irreversible DSC transition for apo human lactoferrin, a double DSC transition for apo human serum transferrin (one reversible) and a broad irreversible asymmetric DSC transition for apo rabbit serum transferrin. FTIR spectroscopy shows that a distinct loss of protein secondary structure occurs at the transition temperatures shown by DSC.

Animals↗

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Bacillus↗

Thermal phase transitions in sheep, rat and rabbit surfactant lipids detected by differential scanning calorimetry.

1. The thermal behaviour of sheep, rat and rabbit pulmonary surfactant lipids was investigated using high sensitivity differential scanning calorimetry (DSC). 2. Phase transitions were evident in the surfactant lipids from all three animals, with the upper limit of the phase transition being 30.1 C in the sheep, 36.8 C in the rat and 36.3 C in the rabbit. 3. The relatively greater fluidity of the sheep surfactant lipids in comparison to those of the rat and rabbit was due primarily to differences in their palmitic acid content.

Animals↗

Direct and indirect calorimetry measurements during the annual growth cycle of juvenile frogs Rana ridibunda P.

A thermodynamic study of the energetic metabolism in juvenile amphibians was conducted over an annual cycle of growth. The total energy flux (delta H = enthalpy variation) was measured with direct microcalorimetry and accounted for the sum of all metabolic pathways. Indirect calorimetry, oxygen consumption measurements (VO2), provides the values for the oxidative metabolism-dependent energy. A comparison between the results of both techniques simultaneously performed, provides for the calculation of the energetic efficiency, which indicates the respective importance of aerobic and anaerobic metabolisms during the annual biological cycle. It is clear that oxidative metabolism does not represent all, nor even a constant percentage of the total energy production of the frog, Rana ridibunda. The ration (Formula: see text) varied between 0.72 and 1.00, according to the season and the feeding or fasting periods. These results are discussed with special reference to the ecophysiology and behaviour of this species.

Acclimatization↗

Influence of collagen denaturation on the chemorheological properties of skin, assessed by differential scanning calorimetry and hydrothermal isometric tension measurement.

The curves obtained for skin samples of different ages and species by hydrothermal isometric tension ("HIT") measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0 degrees C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the "HIT" curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the "HIT" curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both "HIT" and DSC are useful methods to evaluate collagen reticulation in connective tissues.

Aging↗