Enthalpy of denaturation

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. 

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.

The use of differential scanning microcalorimetry for measuring the thermal denaturation of proteins is described in Chapter 17, section Ale. Typically, 0.5-1 mg of protein in 1 mL of buffer, or 0.1-0.2 mg in 0.5 mL with the most sensitive apparatus, is required for an accurate determination of the enthalpy of denaturation. The thermodynamics of dissociation of a reversibly bound ligand may be calculated from its effects on the denaturation curve of a protein.14 The binding of ligands always raises the apparent Tm (temperature at 50% denaturation) of a protein because of the law of mass action the ligand does not bind to the denatured state of the protein, and so binding displaces the denaturation equilibrium toward the native state. 

Fig. 4. Temperature dependence of the specific enthalpy of denaturation of myoglobin and ribonuclease A (per mole of amino acid residues) in solutions with pH and buffer providing maximal stability of these proteins and compensation of heat effects of ionization (see Privalov and Khechinashvili, 1974). The broken extension of the solid lines represents a region that is less certain due to uncertainty in the A°CP function (see Fig. 2). The dot-and-dash lines represent the functions calculated with the assumption that the denaturation heat capacity increment is temperature independent.

Kato, A. et al., Enthalpy of denaturation and surface functional properties of heated egg white proteins in the dry state, J. Food Sci., 55, 1280, 1990b. 

The investigation of maleylated vicilin, the 7-S-globulin from faba beans, revealed the existence of a critical degree of modification for the unfolding of this type of protein, too [57]. While no significant changes in denaturation temperature and enthalpy of denaturation were found up to 63% modification, these values decrease signifi-... 

DSC has also been used to quantify the protein denaturation by heat and is a generally accepted technique for the study of the energetics of protein denaturation (Ibanoglu, 2005 Anandharamakrishnan et al., 2007). It measures the denaturation by measuring the heat capacity of a sample as a function of temperature. The enthalpy of denaturation indicates the amount of native protein that is present in the sample. Higher enthalpy values indicate higher extent of residual protein. DSC also can provide important thermodynamic parameters such as enthalpy and heat capacity change (Bruylants et al., 2005). 

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

The standard-state enthalpy of denaturation (A// ) can be calculated from the slope of the natural logarithm of the equiUbrium constant versus inverse temperature plot (Fig. 12.96) using the van t Hoff equation ... 

When a native protein in solution is heated to a high enough temperature, its polypeptide chain will unfold to become the denatured protein. The temperature at which a large portion of the protein unfolds is called the melting temperature. The melting temperature of a certain protein is found to be 46°C, and the enthalpy of denaturation is 382 kJ/mol. Estimate the entropy... 

Another phase change is the denaturation of a protein from a normal, folded state to a disrupted, unfolded state-just like what happens when an egg is cooked. The Clausius-Clapeyron equation is applicable, but here the "pressure" of the phase is equal to the fraction that is denatured at a particular temperature. Lysozyme, an enzyme that breaks down bacterial cell walls, has an enthalpy of denaturation, AdenW, that is 160.8 kJ/mol. If the denatured fraction of lysozyme, fdeiv is 0.113 at 45.0°C, what is fjen 75.0°C ... 

At 298 K, the enthalpy of denatiu on of hen egg white lysozyme is +217.6 kJ mol and the change in tbe constant-pressure molar heat capacity resulting from denaturation of tbe protein is +6.3 kJ K mol", (a) Estimate the enthalpy of denaturation of the protein at (i) 351 K, the melting temperature of the macromolecule, and (ii) 263 K. State any assumptions in your calculations, (b) Based on your answers to part (a), is denaturation of hen egg white lysozyme alsrays endotbermic ... 

---