Denaturation temperature-induced

In a study of the temperature-induced reversible denaturation of the protein chymotrypsinogen,... 

A major problem in unfolding studies of large proteins is irreversibility. In a study of elastase temperature-induced denaturation, second-derivative FTIR show a distinct loss of several sharp amide V features (dominant /3-sheet components and growth in broadened bands at 1645 and 1668 cm-1 (Byler et al., 2000). These features persisted on cooling, indicating lack of reversibility, a feature common to longer multidomain proteins. A graphic example of this is seen in the triosephosphate... 

In summary, formalin-treated does not significantly perturb the native structure of RNase A at room temperature. It also serves to stabilize the protein against the denaturing effects of heating as revealed by the increase in the denaturation temperature of the protein. However, formalin-treatment does not stabilize RNase A sufficiently to prevent the thermal denaturation of the protein at temperatures used in heat-induced AR methods as shown by both DSC and CD spectropolarimetry. This denaturation likely arrises from the heat-induced reversal of formaldehyde cross-links and adducts, as shown in Figure 15.4 of Section 15.4. Further, cooling formalin-treated RNase A that had been heated to 95°C for 10 min does not result in the restoration of the native structure of the protein, particularly in regard to protein tertiary structure. 

Decrease of velocity with higher temperature Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Figure 5.7). 

Vithayathil et al. (34%). In 0.5 M HC1 at 30° RNase-A undergoes structural alterations which can be detected chromatographically at neutral pH. However, all the products are equally active enzymically, and no reaction would have been detected by assay. At pH 11.0 even more involved structural changes take place quite rapidly. Irreversible alkaline denaturation takes place at higher pH and is very rapid at 13. Here the activity loss is accompanied by marked spectral changes indicating reactions such as / elimination at cystine or serine residues (343). A temperature-induced isomerization at neutral pH has been reported by French and Hammes. This is discussed in a later section on nucleotide binding. 

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. 

The covalent bonding of polysaccharide and protein may be induced by heating a mixture under conditions of low aw, below the denaturation temperature (Dickinson, 1993) these are the most heat and freeze-thaw stable protein-polysaccharide complexes (Stainsby, 1980). 

DNA is stable at high temperatures up to 60-65°C. Raising the temperature induces DNA denaturation, and the two-single strands split up. This can be evidenced by following the OD variation of DNA at 260 nm at increasing temperatures from 20 to 82°C (Marmur and Doty 1959) (see also Figure 12.14). 

Another interesting feature of this extended p-sheet model can be seen in Figure 3b, where we show the effect of temperature on the CD spectrum of Peptide 5. Curiously, when the sample temperature is decreased (to 5 C), the spectrum takes on more of a "random coil character (only 20% p-sheet) but, when the temperature is increased to 85 C, the spectrum exhibits significantly more P-sheet character (38% p-sheet). In other words, high temperatures induce structure and cold temperatures reduce structure. We believe that this represents an excellent example of cold denaturation (19), and it suggests that the thermodynamics of p-sheet formation may be more complex than currently appreciated. 

The multiplicity and the partially unfolded character of the dena-turational states of proteins, especially upon temperature-induced denaturation, hamper any thermodynamical analysis of the data and their interpretation. In particular, evaluation of the conformational entropy terms is actually infeasible. 

Danielewicz-Ferchmin I, Banachowicz E, Ferchmin AR (2006) Properties of hydration shells of protein molecules at their pressure—and temperature-induced native-denatured transition. ChemPhysChem 7 2126-2133... 

In a PEC used to deliver a protein, the latter is often used as one of the polyelectrolyte components of the complex. Examples of PECs in which one of the two polyelectrolyte components is an active substance itself, are complexes of chitosan and insulin. The PEC composed of trimethyl chitosan (TMC) and pegylated TMC (PEG-g-TMC) can be obtained simply by mixing the solutions of TMC and insulin at various mass and charge ratios. These PECs were stable in simulated intestinal fluid at pH 6.8. However, they disintegrated in simulated gastrointestinal fluid at pH 1.2. The PECs also protected insulin from temperature-induced denaturation up to 50 °C and from degradation by trypsin. Based on these results, the authors suggested that polyelectrolyte complexation can be a useful technique for fabrication of insulin delivery systems for oral administration. 

FIGURE 13.13 Typical thermogram for thermally induced protein denaturation obtained hy DSC in which the denaturation temperature Td, the enthalpy of denaturation, and... 

It is apparent that many specific structural factors are important in determining whether a given compound will induce interferon in any test system (DeClercq, et al., 1969). When one substitutes a sulfur for an oxygen, one changes the chemical specificity as well as resistance to hydrolysis and thermal denaturation temperature (T ). 

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