DSC, in which temperature Tis changed linearly with time t, shows the thermal behavior of a system in terms of the heat capacity (dQ/dT), or heat flow (dQ/dt), where Q is evolved heat. Measured for solid protein immersed in organic (plus water) solvents, the DSC curve may represent a multiplicity of processes including water desorption from the protein sample into solvent, protein unfold-ing/denaturation, protein-solvent interactions, and protein destruction and degradation. The DSC curves of solids (which are capable of different transformations [e.g., conformation changes]) are expected to be irreversible.
To minimize the chemical deterioration of the protein, the behavior of protein sample will be considered only at temperatures below 100°C. By changing the water contents of the protein and solvent, it is possible to evaluate the hydration effect on the DSC curve of protein in a suspension. Importantly, temperature-initiated water desorption from protein to the solvent occurred in hermetically closed cell produces a heat effect smeared out in the broad temperature range and does not involve the characteristic peak of heat evolution.
The DSC curve should be recorded for the protein suspension against a some reference material (e.g., solvent). The DSC curve of interest is a difference between the DSC sample curve and the baseline. The latter is evaluated by scanning the volume of solvent as used in the sample experiment against the same reference material. By subtracting the baseline curve from the sample curve, the contribution from the bulk liquid phase is eliminated and the difference is more related with events in the solid phase.
This difference between the DSC sample curve and the baseline is a small difference of great values. Thus, absolute heat capacities are not well-reproducible values. As such, we considered the DSC method mostly as a half-quantitative tool to examine protein interactions in a heterogeneous system. Most attention should be paid to the characteristics of the heat evolution peaks such as peak maximum temperature and peak area. The latter is related to the enthalpy change during the temperature-initiated process. Our main expectation in using the DSC calorimetry was based on the idea that protein-solvent interactions "frozen" partially at ambient temperatures may be seen at higher temperatures. Protocol presented here was applied in refs. 15 and 16 to the DSC measurements of human serum albumin in organic solvents. An earlier example of use of the DSC was given for solid ribonuclease in organic solvents (17). There is a related DSC study of ribonuclease immobilized on Celite in organic solvents (18). Because the immobilization was performed in order to avoid protein aggregation effects, such an immobilized protein should be differentiated from the suspended protein in which aggregation effects may play a significant role in the protein rigidity.
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