Proteins, thermal denaturation

Table 2.2 Thermodynamic and structural parameters of soluble proteins. Thermal denaturation free energy change, AG, under reducing conditions and comparable temperatures for an exhaustive set of monomeric uncomplexed proteins with disulfide bonds and without prosthetic groups or ion coordination [11]. Deviations from the balance relation are measured by Y-(5X + 20) and shown to anticorrelate tightly (R2 = 0.72, Fig. 2.3) with the denaturation free energies...

AI Hohlberg. Kinetics of bean protein thermal denaturation. J Food Process Preserv ii 31-42 (1987). 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Here,. Ai(X) is the partial SASA of atom i (which depends on the solute configuration X), and Yi is an atomic free energy per unit area associated with atom i. We refer to those models as full SASA. Because it is so simple, this approach is widely used in computations on biomolecules [96-98]. Variations of the solvent-exposed area models are the shell model of Scheraga [99,100], the excluded-volume model of Colonna-Cesari and Sander [101,102], and the Gaussian model of Lazaridis and Karplus [103]. Full SASA models have been used for investigating the thermal denaturation of proteins [103] and to examine protein-protein association [104]. 

Like most chemical reactions, the rates of enzyme-catalyzed reactions generally increase with increasing temperature. However, at temperatures above 50° to 60°C, enzymes typically show a decline in activity (Figure 14.12). Two effects are operating here (a) the characteristic increase in reaction rate with temperature, and (b) thermal denaturation of protein structure at higher tem-... 

As with any reaction, temperature has an important effect on the rate of an errzy-matic reaction, albeit that the range of interest is limited. For each enzyme an optimum temperature exists (37 °C for reactions in human beings). At high temperatures the activity decreases due to thermal denaturation of the protein constituting the enzyme. 

Purely thermal denaturation of proteins requires much longer times collagen in moist heat below 120 °C needs 30 min to denature (Meyer et ah, 2005), wheat glutens must be subjected to 200-215 °C of dry heat for 72 min (Friedman et ah, 1987), and as mentioned above, whey proteins require at least 50 °C and 30 min for texturization without the use of extrusion processing. 

It is demonstrated here that extrusion is an effective tool for texturing whey proteins to create new functions for dairy proteins and that thermally denatured WPl is a unique ingredient that can be used in large amounts in nontraditional applications for non-TWPl. This review covers the use of extrusion texturized dairy ingredients in foods however, there are other examples of fhe successful use of this technique along with the product, TWPl in different types of nonfood applications, such as in biodegradable films, and bioplastics. 

Other factors that can impact these constants relate to reaction solution conditions. We have already discussed how temperature can affect the value of kCM and kcJKM according to the Arrhenius equation (vide supra). Because enzymes are composed of proteins, and proteins undergo thermal denaturation, there are limits on the range of temperature over which enzymes are stable and therefore conform to Arrhenius-like behavior. The practical aspects of the dependence of reaction velocity on temperature are discussed briefly in Chapter 4, and in greater detail in Copeland (2000). 

Thermally denatured proteins have been studied for a variety of systems using FTIR and VCD. The resulting high-temperature spectra often reflect the characteristics seen earlier for random coil peptides as well as that seen for the unstructured casein. Particularly the amide I IR bands show a frequency shift to center on a broadened band at 1645-50 cm-1. The amide I VCD loses its distinctive character (Fig. 11) and tends toward... 

Privalov et al. (1989) also reported the temperature dependence of the ellipticity at 222 nm for the proteins studied at various pH values (Fig. 28). At the highest temperature studied (80°C), the 222 nm ellipticity value for the thermally unfolded, acid-unfolded, and Gdm-HCl-unfolded proteins appear to be converging, but show a range of 2000 deg cm2/dmol out of a total of 5000 deg cm2/dmol. (ApoMb is an exception in that, as noted before, the thermally denatured protein is apparently an associated /1-sheet. However, the acid- and Gdm HC1-unfolded forms of apoMb have similar [0] 222 values at 80°C.)... 

Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. 

An excluded-volume random-coil conformation will be achieved when the solvent quality exceeds the theta point, the temperature or denatu-rant concentration at which the solvent-monomer interactions exactly balance the monomer—monomer interactions that cause the polymer to collapse into a globule under more benign solvent conditions. A number of lines of small-angle scattering—based evidence are consistent with the suggestion that typical chemical or thermal denaturation conditions are good solvents (i.e., are beyond the theta point) and thus that chemically or thermally unfolded proteins adopt a near random-coil conformation. 

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. 

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