High-sensitivity Differential Scanning Microcalorimetry Research Articles

Structural and thermodynamic properties of rice starches with different genetic background

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose "tie chains", amylose-lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae ("dangling" chains), as well as amylopectin chains with DP 6-12 and 25-36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.

Effect of high pressure on thermal, structural and osmotic properties of waxy maize and Hylon VII starch blends

Hylon VII, waxy maize starch and their mixtures (1:1, 1:3, 3:1, w/w) were subjected to a high pressure treatment (650 MPa/9 min) in an excess of water (30%). High sensitivity differential scanning microcalorimetry (HSDSC), X-ray, Pulse 1H-NMR spectroscopy, and scanning electron microscopy (SEM) were used to analyse changes in the physico–chemical properties and microstructure of modified starches. High pressure-treated Hylon VII showed a slight degree of granules gelatinisation (GD = 10.8%). Whereas, “pressurised” waxy maize starch manifested complete disintegration of granules (GD = 85.9%) and formed a gel-like structure. The gelatinisation degree of “pressurised” starch mixtures decreased along with an increase in Hylon VII concentration. The starch preparations (650 MPa/9 min) after rehydration revealed low values of a swelling index and mobility of water molecules along with an increase in Hylon VII concentration. The high pressure-treated starches, with the exception of Hylon VII, manifested significant changes in their crystalline structure.

Annealing of normal and mutant wheat starches. LM, SEM, DSC, and SAXS studies

Structure and thermodynamic properties of native and annealed wheat starches with different amylose content (from 1.5% to 39.5%) have been studied by high-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray diffraction (SAXS), light (LM), and scanning electron microscopy (SEM). Starch morphology, the values of the melting cooperative unit, the thickness of crystalline lamellae and the size of amylopectin clusters as well as thermodynamic parameters characterizing surface of the face side in starch crystals were determined. Some suppositions based on different physical approaches are used for a discussion of the results concerning structural reorganization of starches on different levels of macromolecular organization.

Molecular basis of the gelatinisation and swelling characteristics of waxy barley starches grown in the same location during the same season. Part II. Crystallinity and gelatinisation characteristics

Abstract Nine waxy barley samples (grown at the same site during the same season) were investigated to identify those molecular aspects of amylopectin structure and architecture which define the order and gelatinisation characteristics. Using 13C CP-MAS/NMR it was confirmed that the number of double helices within the starches were approximately constant although differences in crystallinity were identified by X-ray diffraction. These differences in terms of amount of crystalline order correlated well with gelatinisation temperatures. The onset (To), peak (Tp) and conclusion (Tc) gelatinisation temperatures were 53.4, 59.2 and 68.1 °C on average with the associated enthalpy (ΔH) of 11.0 and 13.5 J g−1 on a starch and amylopectin basis. Annealing of the starches below To elevated To, Tp and Tc by +11.9, +8.2 and +5.1 °C on average and sharpened the gelatinisation range (Tc−To). Acid hydrolysis after annealing increased To, Tp and Tc (especially Tc) by +2.3, +17.4 and +34.7 °C on average. Annealing in the presence of α-amylase elevated similarly the gelatinisation parameters by +10.2, +7.1 and +2.8 °C for To, Tp and Tc, respectively. Crystalline lamellae lengths were found to be 5.2±0.7 and 6.2±0.4 nm using high sensitivity differential scanning micro-calorimetry and differential scanning calorimetry, respectively.

Light scattering and microcalorimetry studies on aqueous solutions of thermo-responsive PVCL-g-PEO copolymers

Dilute aqueous solutions of thermo-responsive poly(N-vinyl caprolactam)-graft-polyethylene oxide (PVCL-g-PEO) copolymers were studied by light scattering and high sensitivity differential scanning microcalorimetry. These copolymers are double hydrophilic at low temperatures, but become amphiphilic upon heating the solutions above the cloud point temperature of the PVCL segments (TCP). The self-assembly properties of the copolymers are investigated by dynamic light scattering as a function of the temperature, degree of grafting and concentration. It was found that a certain critical polymer concentration is needed for the polymers to form stable aggregates. These structures are expected to consist of a hydrophobic PVCL core, stabilized by a hydrophilic PEO shell. The size of these aggregates increases with the degree of grafting. Microcalorimetry results revealed that the grafting of PVCL with hydrophilic PEO does not influence the phase transition enthalpy of PVCL.

Influence of growth temperature on the structure and thermodynamic parameters of barley starches

Barley starches grown at different temperatures were investigated using high sensitivity differential scanning microcalorimetry and X-ray diffraction. By applying physico-chemical approaches, thickness of crystalline lamellae, thermodynamic and structural characteristics (such as gelatinisation) of cooperative units and parameters characterising thermodynamic properties of crystal surfaces were determined. It was established that a difference of growth temperature experienced by plants during development does not lead to changes in the thickness of amylopectin crystalline lamellae and hence constituent double helix length. The role of defects in structural organisation of native barley starches is discussed. It is suggested that not all fatty acids necessarily form crystalline inclusion complexes.

Thermodynamic and structural properties of starches extracted from potatoes grown at different environmental temperatures

Potato starches grown at different temperatures were investigated using high sensitivity differential scanning microcalorimetry (HSDSC) and Fourier transform infrared spectroscopy (FTIR). By applying physico-chemical approaches, the thickness of crystalline lamellae and the thermodynamic and structural characteristics (such as gelatinisation) of cooperative units and their surfaces were determined. It was established that a difference of growth temperature experienced by tubers during development does not lead to changes in the thickness of amylopectin crystalline lamellae and hence the constituent double helical length. However, the positive correlation established between growth temperature and gelatinisation temperatures was confirmed as being due to optimisation of crystallite structural organisation (at higher temperatures).

The Application of Different Physical Approaches for The Description of Structural Features in Wheat and Rye Starches. A DSC Study

Starches isolated from wheat (Rapoport's variety) at different stages of maturation and from completely maturated rye (Viatka-2 variety) were investigated in excess of water and in 0.6 M KCl using high sensitivity differential scanning microcalorimetry (HSDSC). Applying some physico-chemical approaches, the starch polymorphous structure, the thickness of the crystalline lamellae and the thermodynamic parameters characterizing their surface faces were determined. It has been established that wheat and rye starches belong to the A-type starches. It is suggested that during biosynthesis of granules the formation of the A-type structure is accompanied by an accumulation of crystal defects. This process leads to a decrease in the melting temperature of starches.

The Application of Different Physical Approaches for The Description of Structural Features in Wheat and Rye Starches. A DSC Study

Starches isolated from wheat (Rapoport's variety) at different stages of maturation and from completely maturated rye (Viatka-2 variety) were investigated in excess of water and in 0.6 M KCl using high sensitivity differential scanning microcalorimetry (HSDSC). Applying some physico-chemical approaches, the starch polymorphous structure, the thickness of the crystalline lamellae and the thermodynamic parameters characterizing their surface faces were determined. It has been established that wheat and rye starches belong to the A-type starches. It is suggested that during biosynthesis of granules the formation of the A-type structure is accompanied by an accumulation of crystal defects. This process leads to a decrease in the melting temperature of starches.

Kinetics of gelatin transitions with phase separation: T-jump and step-wise DSC study

The isothermal gelation (or melting) of gelatin after fast cooling (or heating) steps is studied by using high sensitivity differential scanning micro-calorimetry, in order to determine the dependence of the kinetic and thermodynamic parameters upon changes in composition and in temperature. The calorimetric heat flow curves, obtained according to defined temperature profiles, have been fitted with exponential functions (simple exponentials or stretched exponentials for the step-wise and for T-jump experiments, respectively). The gelation process of gelatin alone for t<300 min shows that the characteristic time τ and the fractional exponent are β very sensitive to the concentration of gelatin chains and to the microscopic phase segregation due to the presence of another polymeric component.

Multidomain structure of a recombinant streptokinase. A differential scanning calorimetry study.

The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.

Changes of Thermodynamic and Structural Properties of Potato Starches (Udacha andAcrosil Varieties) during Biosynthesis

Starches isolated from two potato varieties (Udacha, Acrosil) at different stages of maturation were investigated using X-ray diffraction and high sensitivity differential scanning microcalorimetry methods. Applying some physico-chemical approaches, the thickness of the crystalline lamellae and the thermodynamic parameters characterizing their surface faces were determined. The thickness of crystalline lamellae is practically equal to the corresponding value in an amylopectin model proposed by Robin et al. The values of the thermodynamic parameters for the face sides of the starch crystalline lamellae are one order of magnitude smaller than the corresponding values for polyethylene crystals. Some concepts concerning structural changes during biosynthesis if starch potato granules are discussed.

Studies of the Thermal Volume Transition of Poly(N-isopropylacrylamide) Hydrogels by High-Sensitivity Differential Scanning Microcalorimetry. 2. Thermodynamic Functions

We report the first accurate measurements of the partial heat capacity of poly(N-isopropylacrylamide) hydrogels with varying cross-link density. When the cross-link density is increased, the transition broadens and the transition temperature decreases, while the enthalpy, entropy, and heat capacity increment of the transition do not practically change. The transition heat capacity increment is negative, Δtcp = −0.63 ± 0.04 J/g/K. This indicates the formation of a hydrophobic core of the gel upon the transition. The partial heat capacity of polymer network in the gel approaches the partial heat capacity of the unfolded linear poly(N-isopropylacrylamide) at low temperatures, indicating a complete disordering of the gel under these conditions. On the basis of the calorimetric data, thermodynamic functions of the transition were calculated from 0 to 150 °C. They allow one to compare enthalpic and entropic contributions to the stabilization of the collapsed gel. This state is found to be most stable at about 100 °C, and a reswelling transition could be expected only above 150 °C. Contributions of the dehydration of apolar and polar groups as well as residual factors to the transition enthalpy, entropy, and free energy were calculated. The role of apolar dehydration, i.e., of the hydrophobic effect, was not found to be predominant. Apparently, interactions of residues (van der Waals interactions and/or hydrogen bonding) contribute mainly to the stabilization of the collapsed state.

Heat Capacity Values of 1% Aqueous Dispersions for Native Maize Starches with Different Amylose Content

The heat capacities (HC) of 1% aqueous dispersions of native maize starches with different amylose content were determined by using high sensitivity differential scanning microcalorimetry (HSDSC). The application of an additive equation, taking into account the contributions of different ordered structures (B-, B * - and V-types) to the heat capacity of the high amylose maize starches, allows to describe the changes in the heat capacity of the maize starches at an increase in amylose content from 0% to 70%. The heat capacity values per 1% change both of B- and V-type structures in high amylose starches have been determined. Additionally, the calculations showed that at an amylose content in maize starches equal to 0%, the heat capacity values for A- and B * -type structures are, 164 Jmol -1 K -1 and 186 Jmol -1 K -1 , respectively. The relationship between heat capacity values and structural features of maize starches is discussed.

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150°C. All lysozyme samples exhibit a cooperative conformational transition in both solvents occurring between 10 and 100°C. The transition temperatures in glycerol are similar to those in water at the corresponding pHs. The transition enthalpies in glycerol are substantially lower than in water but follow similar pH dependences. The transition heat capacity increment in glycerol does not depend on the pH and is 1.25±0.31 kJ mol −1 K −1, which is less than one fifth of that in water (6.72±0.23 kJ mol −1 K −1). The thermal transition in glycerol is reversible and equilibrium, as demonstrated for the pH 8.0 sample, and follows the classical two-state mechanism. In contrast to lysozyme in water, the protein dissolved in glycerol undergoes an additional, irreversible cooperative transition with a marginal endothermic heat effect at temperatures of 120–130°C. The transition temperature of this second transition increases with the heating rate which is characteristic of kinetically controlled processes. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20–80°C but exceeds it at lower and higher temperatures. It is hypothesized that the thermal unfolding in glycerol follows the scheme: N⇔ho-MG⇒U, where N is a native-like conformation, ho-MG is a highly ordered molten globule state, and U is the unfolded state of the protein.

A comparison of the energetics of annexin I and annexin V

The annexins comprise a family of soluble Ca 2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min −1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t 1/2 = 61.8°C, Δ H cal = 824 kJ mol −1 and Δ C p = 19 kJ mol −1 K −1. These parameters result in a stability value of Δ G D 0 (20°C) = 51 kJ mol −1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded Δ G D 0 (buffer) values of 48, 60 and 36 kJ mol −1 at 20, 12 and 5°C, respectively. These Δ G D 0 values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.

Studies of the Thermal Volume Transition of Poly(N-isopropylacrylamide) Hydrogels by High-Sensitivity Differential Scanning Microcalorimetry. 1. Dynamic Effects

Studies of the Thermal Volume Transition of Poly(<i>N</i>-isopropylacrylamide) Hydrogels by High-Sensitivity Differential Scanning Microcalorimetry. 1. Dynamic Effects

Thermodynamic stability of two variants of xylanase (Xys1) from Streptomyces halstedii JM8.

In a continuation of our earlier study [Ruiz-Arribas, A., Santamaría, R.I., Zhadan, G. G., Villar, E. & Shnyrov, V. L. (1994) Differential scanning calorimetric study of the thermal stability of xylanase from Streptomyces halstedii JM8, Biochemistry 33, 13787-13791], we used high-sensitivity differential scanning microcalorimetry, intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism to study the effect of regional sequence differences on the thermodynamic stability of xylanase (Xys1) from Streptomyces halstedii JM8 (1,4-beta-D-xylanohydrolase, EC 3.2.1.8). Thermal transitions were measured for original xylanase (Xys1S) and two variants. Thermal denaturation of all the xylanases studied revealed two structural domains, each of which, despite its partial irreversibility, follows a two-state thermal unfolding process under our experimental conditions. Both variants were found to exhibit slightly decreased stability, possessing the same activity as the original. The unfolding parameters for each domain of both variants, unlike the situation with wild-type xylanase (see our previous report), fit some correlations obtained for the most compact globular proteins. The values of enthalpy and entropy of unfolding/residue at 383 K were found to be inversely proportional to residual, well-regulated structures in unfolded states.

Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction.

Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t1/2 = 54.7 degrees C (heating rate of 2.34 K min-1), delta H0 = 690 kJ mol-1, and delta Cp = 10.3 kJ mol-1 K-1 which correspondends to a value of delta G0D (20 degrees C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration C1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the delta Gapp versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature delta t1/2 (= 18 degrees C) when the GdnHCl concentration is increased from 0 to 1 M. "Regular" proteins experience an average decrease in the transition temperature of 8 +/- 2 degrees C per 1 M change in the concentration of GdnHCl.

Gel-sol phase transition of poly(3-hydroxybutyrate) in dimethylformamide

The formation of thermoreversible (physical) gels in a moderately dilute solution of a low molecular weight poly(β-hydroxybutyrate), PHB, in dimethylformamide, DMF, has been studied by different techniques in order to provide a preliminary understanding of the molecular processes at the basis of the gelation phenomenon of this polymer. The characterisation of gel phase formation has been carried out with several experimental approaches aiming at elucidating the effect of the thermal history and the kinetics of this process. Scattering data have been taken as a measure of the fraction of gelling crystalline aggregates formed as a function of time and analysed according to the Avrami equation. Characterisation of the gel-sol transition temperature(s) and energetics has been carried out by optical methods (e.g., turbidity) and by calorimetry as a function of temperature. The heat associated with the disruption and the formation of the gel has been measured by high-sensitivity differential scanning microcalorimetry.