Proteins denaturation effects

The red blood cells (RBC) test [12] also investigates the protein denaturing effect of surfactants by using a biological material as substrate, the red blood cells. 

Hypothermia—Indirect cryodestruction Metabolic uncoupling Energy deprivation Ionic imbalance Disruption of acid-base balance Waste accumulation Membrane phase transitions Cytoskeletal disassembly Frozen State—Direct cryodestruction Water solidification Hyperosmolality Cell-volume disruption Protein denaturation Tissue shearing Intracellular-ice propagation Membrane disruption Microvascular Thawed State Direct effects... 

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

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. 

Figure 15.8 (a) Time course of the activity restoration of formalin-treated RNase A during incubation at 50°C (0-2h) and 65°C (2-4h) in TAE buffer, pH 7.0. (b) Time course of the activity restoration of formalin-treated RNase A during incubation at 65°C in TAE buffers of various pH values. All RNase A preparations were freed of excess formaldehyde by dialysis prior to the assay. The RNase A activity was determined with a colorimetric assay using cytidine 2,3,-cyclophosphate as the substrate as described by Crook et al.54 Note that the slopes of the curves decrease with incubation time at 65°C, which is near the denaturation temperature of native RNase A. This loss of activity is likely due to the competing effect of protein denaturation of the recovered RNaseA at this temperature. See Rait et al.10 for details. 

This process of cross-linking does not appear have a major effect on protein secondary structure at room temperature. However, cross-links formed by reactions of formaldehyde with proteins retard, but do not eliminate, protein denaturation that occurs when proteins are heated to a temperature of approximately 70°C or above.7... 

Temperature-sensitive mutations usually arise from a single mutation s effect on the stability of the protein. Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures (usually 37°C), the protein folds and is stable and active. However, at a slightly higher temperature (usually 40 to 50°C) the protein denatures (melts) and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions. 

Protein extraction procedures employing chemicals such as detergents are effective in many instances, but they suffer from a number of drawbacks, not least of which is that they often induce protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even if the chemicals employed do not adversely affect the protein, their presence may adversely affect a subsequent purification step (e.g. the presence of detergent can prevent proteins from binding to a hydrophobic interaction column). In addition, the presence of such materials in the final preparation, even in trace quantities, may be unacceptable for medical reasons. 

In summary, the physiological control of silk protein conversion shows an ingenious balance of activating and inhibiting mechanisms that are dependent on composition and sequence arrangement (Krejchi et al., 1994). Denaturing effects observed in silks appear to be identical to those found in amyloid-forming proteins, and they principally alter the competitive outcome of the hydration of nonpolar and polar residues (Anfinsen, 1973 Dill, 1990 Dobson and Karplus, 1999 Kauzmann, 1959). The key differences to amyloids may lie in the hierarchical level of the structures (Muthukumar et al., 1997) involved in the assembly of silks compared to amyloids. 

Astringents such as tannic acid (home remedy black tea) or metal salts precipitate surface proteins and are thought to help seal the mucosal epithelium. Protein denaturation must not include cellular proteins, for this would mean cell death. Although astringents induce constipation (cf AP salts, p. 166), a therapeutic effect in diarrhea is doubtful. 

The protein(s) is relatively unstable at its true pHopt, and this lack of stability has not been corrected in the pH-activity plot. Thus, the observed pHopt is a compromise of the effect of pH on both catalytic activity (under the assay conditions) and protein denaturation and/or conformation. 

A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 3.20. Protein denaturation, for example, entails a partial or complete alteration of the protein s 3-D shape. This is underlined by the disruption of the intramolecular forces that stabilize a protein s native conformation, viz hydrogen bonding, ionic attractions and hydrophobic interactions. Covalent modifications of protein structure that can adversely effect its biological activity are summarized below. 

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