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.     

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.     

Relaxation Kinetics of Lipid Membranes and Its Relation to the Heat Capacity

We investigated the relaxation behavior of lipid membranes close to the chain-melting transition using pressure jump calorimetry with a temperature accuracy of ∼10^-3 K. We found relaxation times in the range from seconds up to about a minute, depending on vesicular state. The relaxation times are within error proportional to the heat capacity. We provide a statistical thermodynamics theory that rationalizes the close relation between heat capacity and relaxation times. It is based on our recent finding that enthalpy and volume changes close to the melting transition are proportional functions.     

Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.     

Calorimetric studies of the kinetic unfreezing of molecular motions in hydrated lysozyme, hemoglobin, and myoglobin.

Differential scanning calorimetric (DSC) studies of the glassy states of as-received and hydrated lysozyme, hemoglobin, and myoglobin powders, with water contents of < or = 0.25, < or = 0.30, and < or = 0.29 g/g of protein, show that their heat capacity slowly increases with increasing temperature, without showing an abrupt increase characteristic of glass-->liquid transition. Annealing (also referred to as physical aging) of the hydrated proteins causes their DSC scans to show an endothermic region, similar to an overshoot, immediately above the annealing temperature. This annealing effect appears at all temperatures between approximately 150 and 300 K. The area under these peaks increases with increasing annealing time at a fixed temperature. The effects are attributed to the presence of a large number of local structures in which macromolecular segments diffuse at different time scales over a broad range. The lowest time scale corresponds to the > N-H and -O-H group motions which become kinetically unfrozen at approximately 150-170 K on heating at a rate of 30 K min-1 and which have a relaxation time of 5-10 s in this temperature range. The annealing effects confirm that the individual glass transition of the relaxing local regions is spread over a temperature range up to the denaturation temperature region of the proteins. The interpretation is supported by simulation of DSC scans in which the distribution of relaxation times is assumed to be exceptionally broad and in which annealing done at several temperatures over a wide range produces endothermic effects (or regions of DSC scans) qualitatively similar to those observed for the hydrated proteins.     

Calorimetric study of the glass transition of denatured collagen

Temperature dependence of heat capacity of native and denatured collagen samples with different content of bound water (6 divided by 27%) has been studied by DSC method in the temperature range from -50 to 150 degrees C. Heat capacity of denatured samples demonstrates a jump of 0.50 J/g.grad. at temperature Tg, which depends on humidity of the sample. It has been shown that Tg value also depends on the heating rate and thermal history. Annealing at the temperature below Tg produces an additional maximum in the temperature dependence on heat capacity. The magnitude of this maximum, as well as the Tg value increase with the annealing time. It is concluded that these properties of heat capacity reflect glass transition in the denatured collagen.     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D(2)O) and hydrogen oxide (H(2)O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H(2)O by D(2)O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D(2)O and H(2)O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H(2)O by D(2)O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D(2)O turned out to need higher pressures than in H(2)O.     

Successive phase-transition phenomena and phase diagram of the phosphatidylcholine-water system as revealed by differential scanning calorimetry

A completely dehydrated dipalmitoylphosphatidylcholine (DPPC) was prepared with dehydration under high vacuum and at a temperature above its main transition temperature. Thermal analyses on about forty different samples of the DPPC-water system indicated that the main transition temperature decreased stepwise with an increase in the water content to the limiting temperature at 42.6°C, reflecting the thermal behaviors of a total of five endothermic peaks. The pretransition appeared at a water content above 17 g%, and the predominant role of ‘newly incorporated water’ between the bilayers of DPPC molecules at the pretransition was made evident.     

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the P_β′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % ²H₂O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.     

Phase transitions of phospholipid vesicles under osmotic stress and in the presence of ethylene glycol.

The effects of poly(ethylene glycol) (PEG) on the phase transition of phospholipid multilamellar vesicles (MLVs) were investigated by using differential scanning calorimetry (DSC). Main transition temperature (Tm) and the pre-transition temperature (Tp) of neutral phospholipid-, DMPC-1, DPPC- and DSPC-MLVs increased with an increase in PEG concentration. The subtransition temperature of DPPC-MLV also increased with an increase in PEG concentration. These results could be qualitatively explained by enhancement of the lateral packing on the basis of the osmoelastic coupling theory. The pretransition temperature increased faster than the main transition temperature did with an increase in PEG concentration. The increment of Tm depended on the hydrocarbon chain length, the shorter the hydrocarbon chain length was, the larger the increment was. The transition width in the DSC peak was broadened with an increase in PEG concentration. These three above-mentioned effects are the main differences between the effects of the osmotic stress on the phase transition of MLVs and those of hydrostatic pressure. On the other hand, ethylene glycol (EG), which is the monomer of PEG, had a biphasic effect on transition temperature of DPPC-, DSPC-, and DMPC-MLV, reducing Tm and Tp at low concentrations, but increasing Tm and extinguishing pretransition at high concentrations. This is explained by the induction of an interdigitated gel phase at high concentrations of EG, which indicates that EG can easily penetrate into the head group region of the lipid, in contrast with PEG 6K, because EG is small. Temperature-EG concentration phase diagrams for the various PC-MLVs were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.     

Effects of cyclosporine A on biomembranes. Vibrational spectroscopic, calorimetric and hemolysis studies.

Cyclosporine A (CSA)-dipalmitoylphosphatidylcholine (DPPC) interactions were investigated using scanning calorimetry, infrared spectroscopy, and Raman spectroscopy. CSA reduced both the temperature and the maximum heat capacity of the lipid bilayer gel-to-liquid crystalline phase transition; the relationship between the shift in transition temperature and CSA concentration indicates that the peptide does not partition ideally between DPPC gel and liquid crystalline phases. This nonideality can be accounted for by excluded volume interactions between peptide molecules. CSA exhibited a similar but much more pronounced effect on the pretransition; at concentrations of 1 mol % CSA the amplitude of the pretransition was less than 20% of its value in the pure lipid. Raman spectroscopy confirmed that the effects of CSA on the phase transitions are not accompanied by major structural alterations in either the lipid headgroup or acyl chain regions at temperatures away from the phase changes. Both infrared and Raman spectroscopic results demonstrated that CSA in the lipid bilayer exists largely in a beta-turn conformation, as expected from single crystal x-ray data; the lipid phase transition does not induce structural alterations in CSA. Although the polypeptide significantly affects DPPC model membrane bilayers, CSA neither inhibited hypotonic hemolysis nor caused erythrocyte hemolysis, in contrast to many chemical agents that are believed to act through membrane-mediated pathways. Thus, agents, such as CSA, that perturb phospholipid phase transitions do not necessarily cause functional changes in cell membranes.     

Effect of lanthanum ions on the phase transitions of lecithin bilayers.

Interaction of lanthanum ions (La3+) with 1,2 dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) causes an increase in Tc, the temperature of maximal excess heat capacity, and the width of the gel-to-liquid crystalline transition. At a mole ratio of La3+ to DPPC sufficient to remove the hydrocarbon chain tilt angle of DPPC, the changes in the thermodynamic parameters of the pretransition are minor, Tc and the width were unaltered and the enthalpy was reduced by only 10%. This suggests that the change in tilt angle is not a necessary concomitant of the pretransition.     

Glass Transition and Dynamics in Lysozyme�CWater Mixtures Over Wide Ranges of Composition

Differential scanning calorimetry (DSC) and two dielectric techniques, broadband dielectric relaxation spectroscopy and thermally stimulated depolarization currents (TSDC), were employed to study glass transition and water and protein dynamics in mixtures of water and a globular protein, lysozyme, in wide ranges of water content, both solutions, and hydrated solid samples. In addition, water equilibrium sorption isotherms (ESI) measurements were performed at room temperature. The main objective was to correlate results by different techniques to each other and to determine critical water contents for various processes. From ESI measurements the content of water directly bound to primary hydration sites was determined to 0.088 (grams of water per grams of dry protein), corresponding to 71 water molecules per protein molecule, and that where clustering becomes significant to about 0.25. Crystallization and melting events of water were first observed at water contents 0.270 and 0.218, respectively, and the amount of uncrystallized water was found to increase with increasing water content. Two populations of ice crystals were observed by DSC, primary and bulk ice crystals, which give rise to two separate relaxations in dielectric measurements. In addition, the relaxation of uncrystallized water was observed, superimposed on a local relaxation of polar groups on the protein surface. The glass transition temperature, determined by DSC and TSDC in rather good agreement to each other, was found to decrease significantly with increasing water content and to stabilize at about −90 °C for water contents higher than about 0.25. This is a novel result of this study with potential impact on cryoprotection and pharmaceutics.     

Identification of the thermal transitions in potato starch at a low water content as studied by preparative DSC

The aim of this work was to identify the transitions in the complex DSC profiles of potato starch at a low water content. Preparative DSC involves the thermal processing of samples in stainless steel DSC pans in a way that allows their subsequent structural characterization. The low temperature (LT), dual melting (M1–M2), and high temperature (HT) endotherms observed in DSC profiles of potato starch with 16% water were assigned to enthalpy relaxation, melting with preservation of granular identity, and transition of the melted granules into a molecular melt, respectively. Granular melting was accompanied by a strong reduction of swelling capacity. Significant molecular degradation was observed after the HT transition. There is evidence that HT does not represent a true thermodynamic transition, but is due to a volume change in the sample. In contrast to potato starch, maize starch with 16% water gave inhomogeneous samples after processing, presumably because of its low packing density.     

Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.     

Using 31P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using 31P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic 31P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the 31P chemical shift anisotropy (CSA) and spin-spin relaxation times (T2), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.