Differential scanning calorimetry denaturation temperature

Structured proteins have also been investigated by thermal analysis [40,41], denaturing resulting in an endotherm which is readily detected by differential scanning calorimetry (DSC). DSC of recombinant resilin in the swollen state showed no transitions over a wide temperature range (25°C-140°C), further evidence of the absence of any strucmre. This is in contrast to the strucmred proteins wool and bovine serum albumin, which show denamration endotherms at 145°C and 62°C, respectively (Figure 9.6). 

Cross-linking constrains the conformational flexibility of biopolymers and, as a rule, stabilizes their secondary, tertiary, and quaternary structures against the denaturing effects of high temperatures.29 We used differential scanning calorimetry (DSC) to compare the heat-induced conformational transitions of selected RNase A samples that were characterized in Figure 15.2. A brief introduction to DSC is provided in Section 15.15.1 for those readers unfamiliar with this biophysical method. Trace 1 in Figure 15.3a is the heat absorption... 

The thermal unfolding of proteins is best measured by differential scanning calorimetry, which measures the heat absorbed by a protein as it is slowly heated through its melting transition (Figure 17.1). A solution of about 1 mg of protein in 1 mL of buffer and a separate reference sample of buffer alone are heated electrically.6 The additional current required to heat the protein solution is recorded. As the protein denatures, there is a large uptake of heat because the process is highly endothermic. The temperature at the maximum of the peak is... 

A study of two of the most prominent and widespread osmolytes, betaine and beta-hydroxyectoine, by differential scanning calorimetry (DSC) on bovine ribonu-clease A (RNase A) revealed an increase in the melting temperature Tm of RNase A of more than 12 K and of protein stability AG of 10.6 kj mol-1 at room temperature at a 3 M concentration of beta-hydroxyectoine. The heat capacity difference ACp between the folded and unfolded state was significantly increased. In contrast, betaine stabilized RNase A only at concentrations less than 3 M. When enzymes are applied in the presence of denaturants or at high temperature, beta-hydroxyectoine should be an efficient stabilizer. 

The increased thermostability of the disulfide mutant is also shown by denaturation temperature estimated by differential scanning calorimetry (Fig. 12.4). Tm (midpoint temperature in the thermally induced transition from the folded to the unfolded state) of the mutant enzyme is 63.0°C, which is 4.5°C higher than that of the wild-type enzyme (58.5 °C). Under reducing conditions, the Tm of the mutant enzyme is decreased to 50.5 ° C, which is lower than that of the wild-type enzyme, indicating that the disulfide bond formation is required for the thermostability and that the mutant enzyme with free cysteine residues does not have a stable structure like the wild-type enzyme. 

Bell et al. [20] found, via differential scanning calorimetry, that the denatu-ration temperature of lyophilized bovine somatotropin and lysozyme decreased, and therefore stability decreased, with increasing moisture content. The denatura-tion temperature decreased with increasing moisture irrespective of the excipient. The magnitude of the decrease in denaturation temperature was, however, dependent on the type of excipient. 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of denaturation occurs with the immobilized enzyme, which may involve a two-phase process. Immobilization offers more resistance to denaturation at the higher temperature of 60°C where the second phase is prolonged by a factor of three [60]. Differential scanning calorimetry experiments showed that bromelain is an exceptional protease among the cysteine proteases, illustrated by the fact that its thermal denaturation is consistent with an irreversible two-state model [61]. Also, the far UV circular dichroism spectrum of bromelain differs from those of papain and chymopapain and therefore represents a third spectral class within the cysteine proteinase family [62],... 

Fig. 3. Differential scanning calorimetry scans of biomembrane transitions, all obtained with 50% ethylene glycol/water as solvent. (A) A. laidlawii membranes from cells grown in tryptose medium at 37 C (B) lysodeikticus membranes from cells grown in brain heart infusion at 37 C (C) JE. coli K12W945 whole cells grown in minimal salts with glucose at 20 C (D) the same cells as in (C), but scanned after thermal protein (E) rat liver microsomes (F) rat liver In all cases a lower temperature reversible lipid transition is followed by a higher temperature irreversible protein peak. The protein denaturation peaks are featureless in (A), (E), and (F), but show fine structure in (B), (C), and (D). Unlike other organisms, coli after heating shows two lipid transitions and residual reversible protein denaturation, as seen in (D).

The most detailed thermodynamic analysis of protein structure stability is based on differential scanning calorimetry (DSC). In a DSC experiment, the heat capacity Cp of a sample is monitored while heating (or cooling) the sample. Figure 13.13 shows a typical DSC thermogram for heat-induced denaturation of a protein in solution. The thermodynamic observables are the temperature of denaturation (the temperature at half-peak area), the enthalpy change An, d (T involved in the denaturation process (the area under the peak), and the change in the heat capacity A,, dC of the solution (the shift of the baseline). 

Differential scanning calorimetry has been used to investigate changes in the conformation of hen egg-white lysozyme brought about by the combined actions of temperature and denaturants. The effects of a wide range of alcoholic denaturants indicated that hen egg-white lysozyme, whether in solution or in crystalline form, displays structural features that are generally strikingly similar. 

The electron transfer properties of this reaction system provided interesting temperature dependencies, and coupled with differential scanning calorimetry, a model for the denaturation mechanism was proposed to include the bound cyto-... 

Our understanding of the thermotropic behavior of the lamallae lipids is better. Four transitions near 40 C, 70 C, 80 C, and 95 C have been detected by differential scanning calorimetry [52]. The two lowest transitions reflect lipid-water and lipid-lipid interactions because they are reversible and are missing in lipid-extracted stratum comeum. The transition at the highest temperature is irreversible and is believed to reflect protein denatur-ation. Finally, the transition at 80 C is attributed to lipid-protein interaction because it is missing from lipid-extracted stratum comeum. The relevance of these lipid transitions to the permeability properties of the skin will be discussed in Section V. 

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