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.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

The glass transition temperatures of amorphous trehalose-water mixtures and the mobility of water: an experimental and in silico study

Isothermal-isobaric molecular dynamics simulations are used to calculate the specific volume of models of trehalose and three amorphous trehalose-water mixtures (2.9%, 4.5% and 5.3% (w/w) water, respectively) as a function of temperature. Plots of specific volume versus temperature exhibit a characteristic change in slope when the amorphous systems change from the glassy to the rubbery state and the intersection of the two regression lines provides an estimate of the glass transition temperature T(g). A comparison of the calculated and experimental T(g) values, as obtained from differential scanning calorimetry, shows that despite the predicted values being systematically higher (about 21-26K), the trend and the incremental differences between the T(g) values have been computed correctly: T(g)(5.3%(w/w))相似文献   

Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control

A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models.     

The protein-solvent glass transition

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G ≈ 170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry

Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 -hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP—Rf complex. For RBP—FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP—Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed 70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Melatonin strongly interacts with zwitterionic model membranes—evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry

Interactions of melatonin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and melatonin concentration (1-30 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The investigation of the C-H, CO, and PO₂⁻ antisymmetric double stretching modes in FTIR spectra and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, and increasing the dynamics of the system both in the gel and liquid crystalline phases. It also causes significant decrease in the wavenumber for the CO stretching and PO₂⁻ antisymmetric double bond stretching bands, which indicates strong hydrogen bonding The results imply that melatonin locates in the interfacial region of the membrane. Furthermore, in the DSC curve, more than one signal is observed at high melatonin concentrations (24 and 30 mol%), which indicates melatonin-induced phase separation in DPPC membranes.     

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.     

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.     

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.     

Monitoring the kinetics and thermodynamics of interfacial enzymatic catalysis by differential scanning calorimetry

Using phase transition profile as an indicator of thermodynamic property and phase transition heat as the second indicator of the percentage of substrates unhydrolyzed, differential scanning calorimetry has been used to observe in detail the kinetics and thermodynamics of phospholipase A(2)-catalyzed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine large unilamellar vesicle (LUV) hydrolysis. Phase transition profiles show that the original LUV almost completely changes into a novel aggregate at the end of the latency, followed by an abrupt activation of the reaction. The phase transition profiles are asymmetric between the heating and cooling curves, indicating a thermodynamic mesostatic property of the system. The reaction in activated phase follows a single first-order kinetics and all of the substrates in vesicles can be hydrolyzed. All these evidences indicate that the products and substrates can freely exchange between the outer and the inner layers of the vesicles and the membrane of the vesicle in the activated phase is permeable. This permeability favors the exchange of the substrates and products, thus, resulting in the activation of the fast reaction.     

Analysis of IgG kinetic stability by differential scanning calorimetry,probe fluorescence and light scattering

Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life‐threatening diseases. For their successful application in the clinic and a favorable cost‐benefit ratio, the design and formulation of these therapeutic molecules must guarantee long‐term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high‐throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three‐step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8‐anilinonaphthalene‐1‐sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy‐to‐perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

Dielectric spectroscopy study of myoglobin in glycerol-water mixtures

Due to the interest in protein dynamics, there are numerous dielectric relaxation studies of proteins in water and in glass-forming aqueous solvents such as glycerol-water mixtures. In the regime of low frequencies, the inevitable dc-conductivity of such systems limits the resolution of dynamics that are slow compared with the solvent relaxation. Solutions of myoglobin in glycerol/water mixtures of various compositions are measured by dielectric spectroscopy in the frequency range from 10 mHz to 10 MHz. The resolution of low frequency modes is improved by two approaches: electrical ‘cleaning’ and the analysis of the derivative of the real component of permittivity, which shows no direct signature of dc-conductivity. Effects of internal interfacial polarization are also addressed by measuring the same solvents in confinement as well as mixed with glass beads. We find two processes, the structural relaxation of the solvent and the slower rotational mode of the protein, with no indication at even lower frequencies of a dielectric signature of fluctuations associated with protein dynamics.     

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry.

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, delta delta G0d, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol-1 (apparent stabilization) for H124L to -2.8 kcal mol-1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between delta delta G0d and the corresponding values for delta delta Hd and T delta delta S0d, the latter quantities being in most cases much larger in magnitude than delta delta G0d. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.     

N-Myristoylethanolamine-cholesterol (1:1) complex: first evidence from differential scanning calorimetry,fast-atom-bombardment mass spectrometry and computational modelling

The interaction of N-myristoylethanolamine (NMEA) with cholesterol is investigated by differential scanning calorimetry (DSC), fast-atom-bombardment mass spectrometry (FAB-MS) and computational modelling. Addition of cholesterol to NMEA leads to a new phase transition at 55 degrees C besides the chain-melting transition of NMEA at 72.5 degrees C. The enthalpy of the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, vanishing at 80 mol%. The enthalpy of the chain-melting transition of NMEA decreases with an increase in cholesterol; the transition disappears at 50 mol%. FAB-MS spectra of mixtures of NMEA and cholesterol provide clear signatures of the formation of ([NMEA+cholesterol]+) ([NMEA+cholesterol+Na]+). These results are consistent with the formation of a 1:1 complex between NMEA and cholesterol. Molecular modelling studies support this experimental finding and provide a plausible structural model for the complex, which highlights multiple H-bond interactions between the hydroxy group of cholesterol and the hydroxy and carbonyl groups of NMEA besides appreciable dispersion interaction between the hydrocarbon domains of the two molecules.