Dynamic analysis of differential scanning calorimetry data

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.     

Statistical analysis of plasma thermograms measured by differential scanning calorimetry

Melting curves of human plasma measured by differential scanning calorimetry (DSC), known as thermograms, have the potential to markedly impact diagnosis of human diseases. A general statistical methodology is developed to analyze and classify DSC thermograms to analyze and classify thermograms. Analysis of an acquired thermogram involves comparison with a database of empirical reference thermograms from clinically characterized diseases. Two parameters, a distance metric, P, and correlation coefficient, r, are combined to produce a 'similarity metric,' ρ, which can be used to classify unknown thermograms into pre-characterized categories. Simulated thermograms known to lie within or fall outside of the 90% quantile range around a median reference are also analyzed. Results verify the utility of the methods and establish the apparent dynamic range of the metric ρ. Methods are then applied to data obtained from a collection of plasma samples from patients clinically diagnosed with SLE (lupus). High correspondence is found between curve shapes and values of the metric ρ. In a final application, an elementary classification rule is implemented to successfully analyze and classify unlabeled thermograms. These methods constitute a set of powerful yet easy to implement tools for quantitative classification, analysis and interpretation of DSC plasma melting curves.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Theoretical basis for differential scanning calorimetric analysis of multimeric proteins

A new general equation simulating irreversible DSC transitions of multimeric proteins was developed. The equation put forward here is the result of an improved mathematical re-elaboration of the classical Lumry-Eyring models, where no restrictive a priori assumptions are made on the kinetic constraints of the denaturation process, or on the enthalpy of the final denatured state. In order to test the wide applicability of this new effective theoretical tool, a series of DSC transitions were simulated with the aim of determining the effects of all relevant thermodynamic, kinetic or experimental parameters on the shape of DSC profiles. Moreover, the classical equations used widely in DSC investigations for the calculus in both kinetic parameters and changes of molecularity, were studied in the light of the model developed here, highlighting, in each case, their rather limited applicability. The new approach proposed in this article was applied to study the thermal denaturation of an hexameric protein (Glucosamine-6-phosphate deaminase), putting in evidence the practical applicability of the theoretical equations developed.     

A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).     

Thermal porosity analysis of croscarmellose sodium and sodium starch glycolate by differential scanning calorimetry

The aim of the study was to demonstrate the applicability of differential scanning calorimetry (DSC) on porosity analysis for cellulose and starch. Croscarmellose sodium (CCS) and sodium starch glycolate (SSG) were allowed to sorb moisture in 85%, 90%, 95%, and 100% relative humidity (RH) at 40°C for 24 hours. The pretreated samples were then subjected to DSC running temperature ranging from 25°C to −50°C at a cooling rate of 10°C/min. The cooling traces of water crystallization, if present, were transformed to porosity distribution via capillary condensation using Kelvin's equation. The porosity analysis of CCS and SSG was also done using nitrogen adsorption as a reference method. It was found that sorbed water could not be frozen (in cases of 85% and 90% RH) until the moisture content exceeded a cutoff value (in cases of 95% and 100% RH). The nonfreezable moisture content was referred to tightly bound, plasticizing water, whereas the frozen one may be attributed to loosely bound water condensation in pore structure of CCS and SSG surfaces. Not only capillary condensation but also the tightly bound, nonfreezable monolayer water lying along the inner pores of the surface contributed to porosity determination. Good agreement with less than 5% deviation of mean pore size was observed when the results were compared with nitrogen adsorption. The narrower pore size distributions, however, were obtained because of the limitations of the technique. It was concluded that pore analysis by DSC could be successful. Further research needs to be done to account for limitations and to extend the applicability of the technique.     

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.     

Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry

Intracellular ice formation (IIF) plays a central role in cell damage during cryopreservation. We are investigating the factors which trigger IIF in Xenopus oocytes, with and without aquaporin water channels. Here, we report differential scanning calorimeter studies of Xenopus control oocytes which do not express aquaporins. Stage I to VI oocytes (which increase progressively in size) were investigated with emphasis on stage I and II because they are translucent and can also be studied under the cryomicroscope. Measurements were made in 1, 1.5, and 2M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 degrees C/min) cooled. EIF in the 1, 1.5, and 2M EG occurred at -6.4, -7.8, and -8.9 degrees C, respectively. Freezing exotherms of individual stage I-VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I-III) was well below T(EIF) while the later stages (IV-VI) froze at temperatures much closer to T(EIF). Thus, in 1.5M EG, T(IIF) was -21.1, -25, and -26.6 degrees C in stages I-III, but was -17 and -8.5 degrees C for stage IV and V-VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I-III) to a low of only 7% in stage V-VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.     

Study of the thermal behavior of azidohetarenes with differential scanning calorimetry

Thermal properties of azidohetarenes without reactive ortho-substituents, obtained by DSC analysis, were compared with the DSC data of ortho-phenyl and ortho-acyl substituted azidohetarenes, which give ring closure reactions either to indoles or to five-membered heterocycles such as isoxazoles. The reaction temperatures and reaction enthalpies give both information to prepare the reaction conditions and important safety information and were, in addition, used to find out relations between the temperatures or enthalpies with the three different reaction mechanisms.     

Study of strong to ultratight protein interactions using differential scanning calorimetry

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of riboflavin binding protein domain interaction using differential scanning calorimetry

Riboflavin binding (or carrier) protein (RfBP) is a monomeric, two-domain protein, originally purified from hens' egg white. RfBP contains nine disulfide bridges; as a result, the protein forms a compact structure and undergoes reversible three-state thermal denaturation. This was demonstrated using a differential scanning calorimetry (DSC) method [Wasylewski M. (2000) J. Prot. Chem. 19(6), 523-528]. It has been shown that the RfBP complex with riboflavin denaturates in a three-state process which may be attributed to sequential unfolding of the RfBP domains. In case of apo RfBP, the ligand binding domain denaturates at a lower temperature than the C-terminal domain. Ligand binding greatly enhances the thermostability of the N-terminal domain, whereas the C-terminal domain thermostability is only slightly affected and, in case of the examined holo RfBPs, the denaturation peaks of both domains merge or cross over. The magnitude of the changes depends on ligand structure. A detailed study of protein concentration effects carried out in this work allowed to estimate not only the thermostability of both domains but also the strength of domain interactions. The DeltaCp, of denaturation was found for C-terminus and N-terminus of RfBP-riboflavin complex to amount to 2.5 and -1.9 kcal mol(-1), respectively. The calculated domain interaction free energy, DeltaGCN, was estimated to be approximately -1580 cal mol(-1) at 67.0 degrees C. This value indicates that the interdomain interaction is of medium strength.     

南极细菌胞外多糖溶液冻结特性的差示扫描量热研究

采用差示扫描量热法,测定几种南极细菌胞外多糖(简称,EPSs)溶液的结晶、熔融、焓转变以及水合性质等冻结特性,分析了EPSs的浓度和分子量与其抗冻活性的关系.结果表明,在溶液冻结过程中,仅0.25%的Pseudoalteromonas sp.S-15-13 EPSs(分子量,6.2×104Da)可抑制冰核形成,溶液冻结温度较纯水的降低(1.07±0.62)℃;溶液的冻结焓降低说明冰核生长变缓,冰晶形成细小,0.125%的Shewanella sp.5-1-11-4 EPSs(分子量,1.2×103Da)和Moritella sp.2-5-10-1 EPSs(分子量,3.0×103Da)冻结焓分别较纯水的降低17.15%和29.13%,S-15-13 EPSs在0.125%～0.5%的范围内可降低冻结焓,0.125%时冻结焓较纯水的低30%,其不冻水含量为(0.292 ±0.05) g/g.在冰晶熔化过程中,几种EPSs均可降低溶液熔融温度和熔融焓,促进冰晶熔化,使冰晶细小;4.0%的5-1-11-4 EPSs、2-5-10-1 EPSs和0.5% S-15-13 EPSs的熔融温度较纯水的分别降低(2.70±0.15)℃、(2.30±0.39)℃和(4.66±0.42)℃.研究结果阐明EPSs可以通过改变菌体周围水的冻结特性,以抵御冰晶对微生物的损伤,大分子量EPSs对冰晶的抑制作用强于低分子量的.     

Heat killing of bacterial spores analyzed by differential scanning calorimetry.

Thermograms of the exosporium-lacking dormant spores of Bacillus megaterium ATCC 33729, obtained by differential scanning calorimetry, showed three major irreversible endothermic transitions with peaks at 56, 100, and 114 degrees C and a major irreversible exothermic transition with a peak at 119 degrees C. The 114 degrees C transition was identified with coat proteins, and the 56 degrees C transition was identified with heat inactivation. Thermograms of the germinated spores and vegetative cells were much alike, including an endothermic transition attributable to DNA. The ascending part of the main endothermic 100 degrees C transition in the dormant-spore thermograms corresponded to a first-order reaction and was correlated with spore death; i.e., greater than 99.9% of the spores were killed when the transition peak was reached. The maximum death rate of the dormant spores during calorimetry, calculated from separately measured D and z values, occurred at temperatures above the 73 degrees C onset of thermal denaturation and was equivalent to the maximum inactivation rate calculated for the critical target. Most of the spore killing occurred before the release of most of the dipicolinic acid and other intraprotoplast materials. The exothermic 119 degrees C transition was a consequence of the endothermic 100 degrees C transition and probably represented the aggregation of intraprotoplast spore components. Taken together with prior evidence, the results suggest that a crucial protein is the rate-limiting primary target in the heat killing of dormant bacterial spores.     

Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.     

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.     

Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1–23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be α-helical in all the lipid mixtures investigated, except for the two CPPs and [1–23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.