Denaturation, enthalpy

Figure 16.12 The temperature-dependent behavior of the denaturation enthalpy and entropy of ribonuclease (RNase) and myoglobin (Mb) under the assumption that AjjjCp is constant (dashed line) or decreasing with increasing temperature (solid line). Reproduced with permission from P. L. Privalov, Ann. Rev. Biophys. Chem. 18, 47 (1989). 1989, by Annual Reviews http //www.AnnualReviews.org...

The general thermodynamic properties of proteins reported above give rise to several questions What do the asymptotic (at Tx) values of the denaturation enthalpy and entropy mean and why are they apparently universal for very different proteins Why should the denaturation enthalpy and entropy depend so much on temperature and consequently have negative values at low temperature In other words, why is the denaturation increment of the protein heat capacity so large, with a value such that the specific enthalpies and entropies of various proteins converge to the same values at high temperature ... 

It is tempting to suppose that all the denaturation enthalpy at Tx is provided by the disruption of nonpolar contacts, i.e., actually by van der Waals interactions, but that the temperature dependence of enthalpy is determined by hydration of nonpolar groups. The latter is supported by the correlation found between AnCp and the saturation of the native structure by the contacts between the nonpolar groups (Table I). However, this simple model immediately raises two questions Why do proteins with different concentrations of nonpolar contacts have the same denaturation enthalpy values at Tx Is it reasonable to neglect the contribution of hydrogen bonds in the denaturation enthalpy ... 

It was suggested earlier that hydrogen bonds in proteins are the main contributors to the denaturation enthalpy at 7X, whereas the nonpolar contacts determine only the temperature dependence of the denaturation enthalpy (Privalov, 1979). The main argument for this was the observation that proteins that have the same enthalpies at Tx have an almost equal concentration of intramolecular hydrogen bonds, but differ in the concentration of nonpolar contacts (Table I). As is evident, the assumption of the dominant role of hydrogen bonds in the stabilization of protein structure explained the observed temperature convergence of the denaturation enthalpy, if the enthalpy of exposure of nonpolar groups to water is zero at this temperature. This assumption implied that either the enthalpy of... 

Pfeil (1981) concluded that a-lactalbumin is less stable than lysozyme, with a lower thermal transition temperature, lower denaturational enthalpy, lower heat capacity change, and lower Gibbs free-energy change. 

FIGURE 13.14 Dependence of the denaturation enthalpy on the denaturation temperature for (a) lysozyme and (b) a-lactalbumin, both dissolved in 0.05 M phosphate buffer pH 7. (Adapted from Haynes, C.A. and Norde, W., J. Colloid Interface ScL, 169, 313, 1995.)... 

FIGURE 13.15 Denaturation enthalpy entropy and Gibbs energy... 

If the relative activity and denaturation enthalpy of the heat-treated (heated to the specified temperature of l°C/min followed by quenching in ice water) free and fixed enzymes are plotted against the heat treatment temperature, there is good agreement between the activity measurement and the DSC one. Thus, it has been demonstrated that DSC can be used to evaluate the thermal stability of enzyme activity. If the thermal treatment temperature exceeds 60° C, the activity of the free enzyme is quickly lost. Although 30% activity is lost at 70°C, 94% of the original activity is lost at 72.6°C. In contrast, the activity of the fixed enzyme is high. Even when... 

In addition to chemical reactions, the isokinetic relationship can be applied to various physical processes accompanied by enthalpy change. Correlations of this kind were found between enthalpies and entropies of solution (20, 83-92), vaporization (86, 91), sublimation (93, 94), desorption (95), and diffusion (96, 97) and between the two parameters characterizing the temperature dependence of thermochromic transitions (98). A kind of isokinetic relationship was claimed even for enthalpy and entropy of pure substances when relative values referred to those at 298° K are used (99). Enthalpies and entropies of intermolecular interaction were correlated for solutions, pure liquids, and crystals (6). Quite generally, for any temperature-dependent physical quantity, the activation parameters can be computed in a formal way, and correlations between them have been observed for dielectric absorption (100) and resistance of semiconductors (101-105) or fluidity (40, 106). On the other hand, the isokinetic relationship seems to hold in reactions of widely different kinds, starting from elementary processes in the gas phase (107) and including recombination reactions in the solid phase (108), polymerization reactions (109), and inorganic complex formation (110-112), up to such biochemical reactions as denaturation of proteins (113) and even such biological processes as hemolysis of erythrocytes (114). 

The van t Hoff plots for thermal denaturation of proteins are linear in the transition region, thus allowing the enthalpy change (AHm) of unfolding at the transition temperature (Tm) to be estimated. Because of the change in free energy in (AG) = 0 at Tm (reversible process), the entropy of unfolding (ASm) at the transition midpoint can be calculated from ... 

Figure 6 depicts the relationship between the specific enthalpy of denaturation measured for CBH I at each of the pH values used in this study, and the of the principal peak at that pH. The strai t line represents a least>squares best fit to the four experimental data points and the empirically derived intersection point (Reference 2, see Discussion) in the upper right comer. All the values determined in the absence of cellobiose, both those at pH values at which the denaturation exhibits a substantial degree of overall reversibility, and those at which the overall process is completefy irreversible, are in reasonably good agreement with the linear relationship. 

Stabilization by Cellobiose. Figure 6 also shows that when different concentrations of the competitive inhibitor cellobiose are present in the DSC samples at pH 4.8 and pH 8.34, the principal denaturation peaks are displaced to higher temperatures, as indicated by the higher T values shown. The AH values, however, do not follow the trend of increasing enthalpy with increasing that is seen for the data in the absence of cellobiose. Instead, the peak areas in the presence of cellobiose are essentially the same as for the peaks appearing at lower temperatures at these pH values in the absence of cellobiose. 

Figure 6. Specific enthalpy of denaturation for native CBH I, plotted as a function of the overall observed as the enzyme molecule is progressively destabilized by increasing the pH. Dot-centered circles represent the specific enthalpy in the absence of cellobiose the straight line is a linear least-squares best fit to these data points, plus the empirically derived intersection point (reference 2, see Discussion) represented by the crossed circle at upper right. The squares represent enthalpies measured at pH 4.80 and pH 8.34 in the presence of the indicated concentrations of cellobiose.

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