Cross thermal denaturation

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

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. 

Cross-linking protects collagen against proteolysis (Vater et al., 1979) and thermal denaturation (Flandin et al., 1984). 

The 2D NOESY spectra in the aH-aH region, where the NOE cross-peaks in the -sheet region (residues 8-14) can be observed, show that the pressure-dissociated monomer is different from the thermal or urea denatured states. Figure 8 compares the NOESY spectra in the aH-aH region for the native state, the pressure-induced monomer state, the thermally denatured state, and the urea-denatured state. The most important finding is that NOE cross-peaks between a-H of Gln-9 and Arg-13 occur even in the pressure-denatured form, indicating the proximity of these two residues. In contrast, no NOEs were observed between a-Hs of Gln-9 and Arg-13 in the NOESY spectra of Arc repressor at 70°C, nor in the presence of 7 m urea. This observation directly proves that Gln-9 and Arg-13 were no longer close to each other in the thermally or chemically denatured states. 

Two types of enzymes were used to introduce cross-links in protein films peroxidase and transglutaminase. A treatment by horseradish peroxidase in the preparation of films of thermally denaturated soy proteins reduced the elongation at elastic limit and increased the tensile strength. ... 

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

Access to nucleic acid dendrimers is initiated by a zip-fastener like dissociation of the DNA double strand by heating. The double strand separates into the two individual strands by thermal motion (denaturation). Subsequent association, hybridisation of complementary sequences, is followed by stepwise cross-linking to form DNA dendrimers, which can contain up to two million oligonucleotide-end group strands (Fig. 8.19). The latter can be labelled with fluorescence or radioactive markers. 

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