A phase, in a non-reacting system, is known to be stable Determination of limit of stability profiles for liquefied natural gas: a thermodynamic approach On this premise, the stability limit determination method by the Helmholtz stability criterion was Stability limit Phase envelop LNG Spinodal Binodal Equation of state. Nov 12, Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total In the present communication using thermodynamic and structural approach, we . We could not go beyond pH as Ficoll becomes unstable or gets. Coy, Daniel Charles, "Visualizing thermodynamic stability and phase equilibrium through computer graphics .. Substance by Means of a Surface" , J. Willard Gibbs proposed the use of the . Klein penetrates beyond the science to present . Having stated the criteria for phase equilibrium, Gibbs then identified the.
It is interesting that all the Gly mutations have similar effects; in all cases holo-SOD is enthalpically destabilized, whereas apo-SOD is entropically destabilized.
Thus, the changes appear to be governed by the removal of the glycine, rather than by the specific characteristics of the substituted residues. Other studies have found that changes in thermodynamic stability caused by mutation tend to follow structurally based statistical preferences for amino acids; however, quantitative interpretation of changes in entropy and enthalpy upon mutation have proven very difficult to rationalize, because of complex effects that may occur in the native and in the denatured states for the protein and for the associated solvent The same is true here.
One might expect that similar arguments would apply to the apoprotein, and so it is perplexing why in fact opposite effects are observed, i. The explanation for this may be related to the very different temperatures where stability changes were measured for holo and apo, i. The metals may also significantly affect entropy and enthalpy changes. In other proteins, comparable mutations involving glycines resulted in similar changes in stability, because of either enthalpy or entropy changes 65 — Further study is clearly still required to understand the molecular basis for changes in stability caused by apparently small changes such as residue substitution or metal binding.
We note, however, that because Gly is relatively rigid in SOD 662mutations may be expected to be more strongly destabilizing compared with mutations at more mobile positions This suggests that the least stable mutants may have the greatest propensity to aggregate.
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One of the main hypotheses for disease mechanism in mutant SOD-associated fALS is increased toxic aggregation of mutant protein 8 Previous studies have reported evidence that more destabilizing mutations have increased tendency to aggregate modulated by effects on net charge of proteinand this may be associated with shorter disease duration 2057 ; however, not all fALS mutations destabilize the apoprotein Thus, the factors underlying aggregation are not yet well understood, and other forms of SOD than apo may also have a role in aggregation in disease.
In vitro measurements of aggregation can be problematic because aggregation may occur very slowly, as may also be the case in vivo. Conditions that decrease protein stability, chemically or by increased temperature, have been used to bring in vitro measurements of aggregation into an experimentally tractable time scale 20 Here we establish a novel approach for measuring aggregation of mutant SOD, which appears to be reversible or constant with time, using DSC.
It is intriguing that the aggregation may be reversible or constant upon rescanning samples and that light scattering measurements as a function of temperature suggest that the aggregates are small, because evidence is mounting that small aggregates may be the toxic species in disease 8. Given that a general correlation between protein and fALS disease characteristics has still not been identified, it is important to further characterize the effects of mutations on holo- and apoprotein.
In this study, we have established a calorimetric method for measuring the effects of mutations on both thermodynamics and aggregation of apo- and holo-SOD. These types of measurements may ultimately contribute to understanding the molecular basis for ALS and to developing urgently needed ALS treatments. In this paper, the special emphasis has given on the working principle, methods and application of the thermodynamic techniques used namely DSC and ITC used frequently in food chemistry.
Differential Scanning Calorimetry Differential Scanning Calorimetry, which measures heat capacity as a function of temperature, is an well-established thermal analysis technique that detects and monitors thermally induced conformational transitions and phase transitions as a function of temperature [ 1920 ].
During temperature scanning, depending on the complexity of the material, many peaks or inflection points one to several reflecting the thermally induced transitions can be observed. The direction of the peak corresponds to the nature of the transition, being heat absorbing endotherms or heat releasing exotherms.
An Overview on the Thermodynamic Techniques used in Food Chemistry
While melting of solids and denaturation of proteins display endotherms, crystallization of carbohydrates and aggregation of proteins manifest themselves as exotherms. DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference are measured as a function of temperature.
Both the sample and reference are maintained at nearly the same temperature throughout the experiment. Generally, the temperature program for a DSC analysis is designed such that the sample holder temperature increases linearly as a function of time.
The reference sample should have a well-defined heat capacity over the range of temperatures to be scanned. The basic principle underlying this technique is that, when the sample undergoes a physical transformation such as phase transitions, more or less heat will need to flow to it than the reference to maintain both at the same temperature.
Whether more or less heat must flow to the sample depends on whether the process is exothermic or endothermic. By observing the difference in heat flow between the sample and reference, differential scanning calorimeters are able to measure the amount of heat absorbed or released during such transitions. Thus the result of a DSC experiment is a curve of heat flux versus temperature.
DSC can be used to measure a number of characteristic properties like crystallization temperature of solids, oxidative stability of samples, used in the pharmaceutical and polymer industries etc. The temperatures for the endothermic and exothermic transitions and the heat involved in such transitions are measured using a calorimeter.
Inflection points are indicative of glass transitions; that is, transitions from a glassy to rubbery state. In this context, it would be quiet unjustified if the older technique Differential Thermal Analysis DTA is not mentioned under discussion. In this technique it is the heat flow to the sample and reference that remains the same rather than the temperature.
When the sample and reference are heated identically, phase changes and other thermal processes cause a difference in temperature between the sample and reference. DSC measures the energy required to keep both the reference and the sample at the same temperature whereas DTA measures the difference in temperature between the sample and the reference when they are both put under the same heat.
DTA instrument can be used at very high temperatures and in aggressive environments where DSC instrument may not work. The basis for thermodynamic study of food materials is that the relevant initial and final states pre-processing and post processing states can be defined and the energetic and structural differences between these states can be measured using calorimetric instrumentation. To this end, calorimetry can be used to evaluate the effect of other physical and chemical variables by comparing the thermo grams of the materials before and after exposure to the variable outside the calorimetry [ 23 ].
However, in most food processing food ingredients are mixed or diluted with a liquid water, milk or with a powder sugar, salt, yeast. For simulation of such transformations and interactions, the limited volume and the lack of in situ mixing constitute the major drawbacks of the DSC technique. Brief instrumental procedure of DSC Experimental set up of a differential scanning calorimeter is represented in Figure 1 A.
The buffer scans were repeated till reproducible and on cooling, the sample cell is rinsed and loaded with sample. The DSC thermograms of excess heat capacity versus temperature plots are analyzed using a variety of softwares like Origin 7.
This calorimetrically determined enthalpy is model-independent and is thus unrelated to the nature of the transition. The temperature at which excess heat capacity is at a maximum defines the transition temperature Tm. Specifically, it is assumed that the transition from the ordered, low temperature form to the disordered, high temperature form passes through no thermodynamically significant intermediate states twostate assumption ; that is, there is no partial unfolding of the protein in the denaturation pathway.
Isothermal Titration Calorimetry Most biological processes involve one or more binding events. The types of binding reactions are varied and include, but are not limited to, assembly of protein subunits into functional enzyme complexes, formation of enzyme-inhibitor complexes, formation of proteinnucleic acid complexes, enzyme-substrate binding, and enzymecofactor binding.
These binding processes can be described in terms of the standard thermodynamic parameters. A predictive understanding of the binding process can be achieved by measurement of the binding thermodynamic parameters. All of the binding processes enumerated above are amenable to analysis by some variation of ITC experiment [ 25 ]. Because the binding sites for small molecules to proteins tend to be well defined and small in number, and because most of the binding reactions involve a nonzero enthalpy change, protein -small molecule interactions frequently are particularly well suited to examination by isothermal titration calorimetry.
Other types of complex equilibria are beyond the scope of this paper. An Isothermal titration calorimetry instrument consists of two identical cells composed of a highly efficient thermal conducting material Hasteloy surrounded by an adiabatic jacket.
Heaters located on both cells and the jacket is activated when necessary to maintain identical temperatures between all components.
In an ITC experiment, the macromolecule solution is generally placed in the sample cell. The reference cell contains buffer or water minus the macromolecule.
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This signal directs the feedback circuit to activate the heater located on the sample cell. This represents the base line signal.
The direct observable measured in an ITC experiment is the time -dependent input of power required to maintain equal temperatures in the sample and reference cell. During the injection of the titrant into the sample cell, heat is taken up or evolved depending on whether macromolecular association reaction is endothermic or exothermic. For an exothermic reaction, the temperature in the sample cell will increase and the feedback power will be deactivated to maintain equal temperatures between the two cells for endothermic reactions, the reverse will occur meaning the feedback circuit will increase power to the sample cell to maintain the temperature.
The heat absorbed or evolved during a calorimetric titration is proportional to the fraction of bound ligand. For the initial injections, all or most of the added ligand is bound to the macromolecule, resulting in large exothermic or endothermic signals depending on the nature of association.
As the ligand concentration increases the macromolecules becomes saturated and subsequently less heat is evolved or absorbed on further addition of titrant as presented in Figure 1. All the solutions used for ITC experiments are degassed prior to use under vacuum mbar, 8 min.
In case of ligand-protein and complexation, degassed ligand solutions are injected from the rotating syringe rpm into the isothermal sample chamber containing 1. Corresponding control experiments to determine the heat of dilution of ligand into buffer and food component into buffer are also performed.