Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions

The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 degrees C and T=67 degrees C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.     

水稻谷胱甘肽磷脂氢过氧化物酶的热稳定性研究

谷胱甘肽磷脂氢过氧化物酶是唯一能够直接还原生物膜上脂类过氧化物的过氧化物酶．本文利用圆二色光谱（CD）、内源荧光光谱和差示扫描量热仪（DSC）研究了温度对谷胱甘肽磷脂氢过氧化物酶（OsPHGPx）活性及其构象变化的影响．在温度为 20－27.5℃ 期间,随着温度的逐渐升高,OsPHGPx 的活性逐渐上升,到 27.5℃ 时达到最大值;在 27.5－45℃ 时,随着温度逐渐升高,其活性迅速下降;当温度超过45℃时,其活性完全尚失. 在20－40℃,CD 光谱、内源荧光光谱和 DSC 均没有发生明显变化,暗示OsPHGPx 的结构基本保持完整;在 40－55℃,CD 光谱显示该酶二级结构发生去折叠;内源荧光光谱的变化暗示该酶三级结构发生去折叠．其中在40－45℃,DSC显示该酶可能存在两个去折叠中间体．当温度超过 55℃ 时,整个酶的构象不再发生变化,呈现去折叠状态．     

F-actin has a very high calorimetric unfolding enthalpy

The thermal unfolding of F-actin was studied using differential scanning calorimetry. Heat denatures F-actin in two steps. The first is endothermic and corresponds to the unfolding of the peptide chain, while the second is exothermic and is due to the aggregation of the unfolded molecules. The aspect of the thermogram is influenced by the concentration of the protein. For concentrations around 1mg/ml, the steps are superimposed, while the two steps are separated at very low concentrations. It thus becomes possible to estimate the calorimetric enthalpy for the unfolding step. The enthalpy of unfolding is 64 MJ/mol, or 1400 J/g. This value is considerably higher than those mentioned in the literature for the denaturation of actin and other proteins, which are in the range of 25-30 J/g. The large amount of energy required to unfold the molecule of F-actin could be an adaptation of its role as a protein that transmits forces, and consequently must be very resistant to mechanical constraints.     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

Elastic Protein-Based Polymers a Step Towards Plasticity: Thermal Stability of Glu-Containing Co-Polypeptides as Analyzed by Differential Scanning Calorimetry

Plastic protein-based polymers with the same conformational potential, but with different degree of thermal stability have been synthesized and thermally characterized by differential scanning calorimetry to provide the conception of behavior of thermoplasticity. Dramatic increase in the temperature between melting and decomposition transitions has been observed, upon inclusion of glutamic acid residue into the hydrophobic sequence of FVGVP. Glu-containing co-polymers of IVGVP showed a markedly different behavior by exhibiting exothermic crystallization transition before melting shows the typical thermoplasticity. Secondary structure in trifluoroethanol for all the polymers show, a well behaved α-helix as evident from the circular dichroism studies, in association with a significant amount of random structure contributes to extended stability.     

Thermal analysis of marginal conditions to facilitate cryopreservation by vitrification using a semi-empirical approach

This study aims at the thermal analysis of marginal conditions leading to cryopreservation by vitrification, which appears to be the only alternative for indefinite preservation of large-size tissues and organs. The term “marginal conditions” here refers to cooling rates in close range with the so-called critical cooling rate, above which crystallization is avoided. The analysis of thermal effects associated with partial crystallization during vitrification is associated with the coupled phenomena of heat transfer and kinetics of crystallization. This study takes a practical, semi-empirical approach, where heat transfer is analyzed based on its underlying theoretical principles, while the thermal effects associated with partial crystallization are taken into account by means of empirical correlations. This study presents a computation framework to solve the coupled problem, while presenting a proof-of-concept for DP6 as a representative cryoprotective agent. The thermal effects associated with crystallization at various relevant cooling rates are measured in this study by means of differential scanning calorimetry. Results of this study demonstrate that, due to the thermal effects associated with partial crystallization, the cooling rate at the center of a large organ may lag behind the cooling rate in its surroundings under some scenarios, but may also exceed the surroundings cooling rate in other scenarios, leading to counter-intuitive effects associated with partial crystallization.     

Thermal study of simple amino-alcohol solutions

Widely regarded as the most promising approach to long-term cryopreservation of organs for transplantation, vitrification is a process where liquid is transformed into a disordered solid state free from crystals, known as the amorphous state. The vitreous state is obtained by rapid cooling to cryogenic temperatures in the presence of antifreeze substances called cryoprotectants, such as polyalcohols, which are known to be very good vitrification agents. This work reports on the thermal properties of a new class of compounds, the amino-alcohols, studied for its similarity to the structure of the equivalent polyalcohols. We studied by differential scanning calorimetry the glass-forming tendency and stability of the amorphous state for de-ionized water solutions containing 2-amino-1-ethanol and 3-amino-1-propanol at the concentrations of 35%, 40%, 43%, and 45% (w/w). A comparison is made with previous results obtained by Mehl [Cryobiology 27 (1990) 687-688] on the same compounds under different experimental conditions. The results are also compared with those obtained by Boutron [Cryobiology 30 (1993) 87-97] for the corresponding dialcohols. A further comparison is made with a few results obtained for the 1-amino-2-propanol and the 2-amino-1-propanol tested under the same conditions.     

使用差示扫描量热仪测定抗冻蛋白热滞活性方法的研究

抗冻蛋白因具有独特的抗冻活性而被研究者广泛关注。但是,目前抗冻活性的检测没有一个标准的、统一的检测方法,这严重制约了该方面的研究进展。作者详细研究了采用差示扫描量热仪测定样品热滞活性的方法,并对该方法的稳定性、专一性和精密度进行评价。结果显示,采用差示扫描量热仪测定样品的热滞活性具有较高的稳定性、重复性和精密度。因此,差示扫描量热仪法可以作为一种通用的方法进行抗冻蛋白热滞活性的检测。     

Differential scanning calorimetric studies on bovine serum albumin IV. Effect of anionic surfactants with various lengths of hydrocarbon chain

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (DSC) have been made at pH 7 on the complexes of BSA and a series of sodium alkyl sulfates. Alkyl sulfates used were sodium decyl sulfate (SDeS), sodium octyl sulfate (SOS), sodium hexyl sulfate (SHS) and sodium ethyl sulfate (SES). Results obtained were compared with those on the system BSA-sodium dodecyl sulfate (SDS) studied previously. Two peaks P 1 and P2 existed in the DSC curve of BSA. These peaks originate in the heat-induced transition of BSA. The pattern of DSC curve changed with the amount of the ligand added, i.e. with the molar mixing ratio ligand/BSA (1). The change for systems BSA-SDeS, BSA-SOS and BSA-SHS was qualitatively the same as that for the system BSA-SDS (2). Interestingly, SES, which is not a surfactant, interacts with BSA. The change for the system BSA-SES was qualitatively the same as that for the system BSA-Na₂SO₄. All alkyl sulfates suppressed the heat-induced transition at lower concentrations. A linear relationship was obtained for the plots of log(D/A)₁ versus log CMC, where (D/A)₁ is the molar mixing ratio of anionic surfactant (D) to BSA (A) at which the most heat-stable complex is formed. This suggests that the hydrophobic force has a serious effect on the formation of heat-stable complexes.     

江浙蝮蛇毒酸性与碱性磷脂酶A_2溶液构象变化的差示扫描量热法研究

运用差示扫描量热法,在不同ｐＨ值的缓冲溶液内和各种浓度的碱土族氯化物溶液内,研究了来自江浙蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｈａｌｙｓＰａｌｌａｓ）毒的酸性与碱性磷脂酶外Ａ２（ＰＬＡ２）的热变性过程。得到表征这两种酶溶液构象变化的热力学参数。依据这些参数研究了两者的溶液构象及其变化。在ｐＨ４．５以下,分子净荷正电的这两种酶在溶液中不形成可热致伸展的有序构象;ｐＨ高于４．５时,Ａｓｐ和Ｇｌｕ的侧链羧基以负离子形式存在有利于有序构象的稳定。Ｈｉｓ是决定ＰＬＡ２活力和热稳定性的重要残基。磷酸根离子和这两种酶有结合作用而降低有序构象的热稳定性。碱土族阳离子除和这两种酶结合外,还以依赖于离子强度的方式复杂地影响酶的溶液构象,但其作用不完全是静电性的,是或多或少地随离子的不同而不同的。计算给出酸性ＰＬＡ２的△Ｈｃｄ．     

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C_25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85 °C to 98 °C, only 10% of entrapped calcein was released. After 10 h at 90 °C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60 °C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C_25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C_25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65 °C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C_25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0 °C to 40 °C.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation

Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer ΔH_c/ΔH_v ratio is close to 4 for the first transition and ∼2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at ∼3–4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Thermal, Mechanical, and Molecular Relaxation Properties of Frozen Sucrose and Fructose Solutions Containing Hydrocolloids

Model frozen systems formulated with 20wt% sucrose or fructose and with the addition of 0.3 or 0.5wt% of xanthan gum (XG), guar gum (GG), locust bean gum (LBG), or a 50wt% mixture of XG and LBG were studied by differential scanning calorimetry, dynamic mechanical analysis, and ¹H-pulsed nuclear magnetic resonance. Melting onset of either the sucrose or fructose model systems was not affected by the addition of hydrocolloids. As expected, ice content was lower in fructose than in sucrose systems. Addition of hydrocolloids had no effect on ice content, except when the blend of XG and LBG was added to the fructose system, where ice content was significantly diminished. Hydrocolloids decreased molecular mobility for both frozen sucrose or fructose solutions, especially for the addition of XG/LBG blend. Relaxation times and storage modulus of the frozen systems with added hydrocolloids were significantly lower than the control frozen sugar solutions.     

A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates

The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30-75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.     

Observation of complex thermal transitions for mixed micelle solutions containing alkyldimethylphosphine oxides and phospholipids and the accompanying cloud points

The thermal properties of various mixtures of two nonionic surfactants, decyldimethylphosphine oxide (APO10) and dodecyldimethylphosphine oxide (APO12) and two phospholipids, dimyristoylphosphatidyl choline (DMPC) and dipalmitoylphosphatidyl choline (DPPC), were examined by differential scanning calorimetry at various mole fractions. The addition of APO12 to DMPC multilamellar vesicles lowered the temperature of the main transition, produced considerable broadening, and eliminated the pre-transition. Phase separation, as evidenced by the existence of a cloud point, T(cp), occurred when the mole fraction of APO12, with respect to DMPC was 0.58 and above. A small abrupt increase in heat capacity was observed at, or slightly above, the cloud point of APO12 and all mixed micelle solutions. It appeared that mixed micelles coexisted with mixed bilayers when the mole fraction was between 0.58 and 0.75 and perhaps as low as a mole ratio of 0.32. All of the mixtures, except APO12/DMPC, exhibited a clear endotherm below the temperature corresponding to the cloud point, which likely reflects the growth in micellar size. Overlapping chain length dependent endothermic peaks, perhaps resulting from reorganization and/or continued association of the micelles, were observed above the cloud point for all of the mixtures except for APO10/DMPC solutions. However, solutions of mixed micelles consisting of APO10/DMPC with mole fractions of surfactant between 0.81 and 0.93 portrayed a broad unidentified exotherm of about 2+/-1 kcal/mol, which was centered nearly 10-20 degrees C above the cloud point.     

Thermal properties of agave fructans (Agave tequilana Weber var. Azul)

Thermal properties of agave (A. tequilana Weber var. Azul) at different water contents were investigated. HP-TLC results showed a complex mixture of mono-, di-, oligo, and polysaccharides in agave fructans samples. The thermal decomposition temperatures were observed below to 200 °C. Modulated-differential scanning calorimetry studies showed a glass transition and a relaxation enthalpy processes in agave fructans. Samples with the highest moieties of monosaccharides showed the lower glass transition temperatures (Tg). The moisture sorption isotherm of agave fructans was determined at 20 °C and fitted to the GAB model. Gordon-Taylor equation was used to fit the Tg experimental data as a function of water content. Agave fructans was found to be an amorphous material. At low water activity (a_w) values (<0.4), agave fructans remained in a powdered amorphous state; and at intermediate a_w (0.4-0.75) collapsed and caked; and at high a_w (>0.75) changed in a highly viscous liquid-like solution.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.     

Triglycerides obtained by dry fractionation of milk fat: 2. Thermal properties and polymorphic evolutions on heating

The crystallographic and thermal properties of milk fat and fractions were investigated on heating using the coupling of synchrotron X-ray diffraction with differential scanning calorimetry. We showed that re-crystallisations occurred during the heating of the stearin and the olein fractions with the formation of a β′ 2L (41.1-42.6 Å) structure and a β′ 3L (66 Å) structure, respectively. By creating a quantified solid-liquid phase behaviour versus temperature diagram, the amount of the solid and liquid phases and the relative proportion of each of the crystalline structures within the solid phase were determined.