Heat capacity denaturation

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

Results from thermal denaturation and heat capacity studies have shown that the proteins are not necessarily completely unfolded in this process. The volume observations also suggest that the denatured state is not one in which all hydrophobic groups are exposed to water. But the results can also be understood from the effect of close polar and electrostatic groups interacting with the water structure surrounding the hydrophobic groups. The volume change is heavily... 

Figure 4. Deconvolution of the denaturational endotherm for native CBHI at pH 4.80. Circles represent experimental values for differential heat capacity the solid curves represent the overall best fit model and the two sequential component transitions that contribute to the overall fit (See text).

We cannot answer the question posed by Anfin-sen s hypothesis. Does the native state have a minimum value of the Gibbs energy Nevertheless, it is observed that proteins usually behave as if folded, unfolded forms are in a true thermodynamic equilibrium, and that this equilibrium is attained rapidly. The difference AG between a folded and a denatured protein is only 21-63 kj mol-1, which shows that folded proteins are only marginally more stable than are unfolded polypeptide chains.645 The value of AG of unfolding as a function of temperature T is given by Eq. 29-13, where AH(T) and ACp are the changes in enthalpy and heat capacity upon unfolding.645 646... 

C and Tm = -19°C and +57°C, i.e., the protein is denatured by either cooling below 18°C or heating above 57°C, a behavior that is common for many proteins. Cold denaturation is observed whenever the unfolded state has a higher heat capacity than does the folded state.647... 

Reversibility of heat denaturation is checked both optically, by the ellipticity, and ca-lorimetrically, by the heat capacity and enthalpy. At acidic pH, the reversibility is about 100% upon heating up to 100 °C. At neutral pH, reversibility is 90% upon heating to 85 °C, and decreases upon heating to higher temperatures. 

The Calorimetrically Obtained van t Hoff Enthalpy In a manner analogous to that used to obtain the van t Hoff enthalpy from the fractional change in the optical absorbance, one can use the temperature dependence of the fractional enthalpy as a function of temperature to determine an effective enthalpy. We will adopt the notation to represent the total enthalpy associated with the denaturation transition. It can be obtained from an integration of the excess heat capacity, corrected for the baselines, as discussed before ... 

In all globular proteins studied, a significant increase in the heat capacity of the denatured protein relative to the native state has been observed in the vicinity of the denaturation transition. (This quantity is represented in... 

A study of two of the most prominent and widespread osmolytes, betaine and beta-hydroxyectoine, by differential scanning calorimetry (DSC) on bovine ribonu-clease A (RNase A) revealed an increase in the melting temperature Tm of RNase A of more than 12 K and of protein stability AG of 10.6 kj mol-1 at room temperature at a 3 M concentration of beta-hydroxyectoine. The heat capacity difference ACp between the folded and unfolded state was significantly increased. In contrast, betaine stabilized RNase A only at concentrations less than 3 M. When enzymes are applied in the presence of denaturants or at high temperature, beta-hydroxyectoine should be an efficient stabilizer. 

Mikulecky, P. J., and Feig, A. L. (2004). Heat capacity changes in RNA folding Application of perturbation theory to hammerhead ribozyme cold denaturation. Nucleic Adds Res. 32, 3967-3976. 

Fig. 8. Experimental ( — ) and theoretical heat capacity functions for the thermal folding/unfolding transition of phosphoglycerate kinase at pH 6.5 in the presence of 0.7 M GuHCl. The heat denaturation transition is characterized by a single peak, whereas the cold denaturation displays two peaks corresponding to the independent unfolding of the N and C domains. The experimental curve has been published before (Griko et al., 1989). As discussed in the text, the theoretical curve does not represent the best fit to the experimental data, but rather the calculated curve using structural information in conjunction with thermodynamic information for elementary interactions. [Reprinted from Freire et al. (1991).]...

The thermal stability of metmyoglobin and apomyoglobin has been extensively studied under different solvent conditions (Privalov et al., 1986). In particular, it was shown that at low pH values both the heat and cold denaturation peaks are clearly visible in the calorimetric scans. Figure 10 shows the excess heat capacity function for apomyoglobin predicted by the hierarchical partition function and the thermodynamic parameters described above. In order to simulate the experimental curve obtained at pH 3.83 (Privalov et al., 1986), the protonation of five specific histidine residues on unfolding was... 

Fig. 10. Predicted excess heat capacity function versus temperature for myoglobin. The curve simulates the experimental curve obtained at pH 3.83 by Privalov et al. (1986). Under those conditions both the cold and heat denaturation curves can be studied experimentally. The predicted values are Tm,cold = 4°C Tm>heat = 58°C A// = 59 kcal mol-1 ACp = 2.45 kcal K-1 mol-1. The experimental values are Tm>coid = 3°C Tm>heat = 57.5°C AH = 53 kcal mol-1 ACP = 2.5 kcal K-1 mol-1 (Privalov et al., 1986). [Reprinted from Freire and Murphy (1991).]...

Cf heat capacity of denatured state at constant pressure... 

AgCp change in heat capacity at constant pressure from native to denatured... 

Fig. 1. Partial specific heat capacity of sperm whale metmyoglobin in aqueous solutions with different pH values in the temperature range in which heat denaturation takes place. The observed heat capacity peak corresponds to the heat absorption upon protein denaturation that also results in a significant heat capacity increase A°CP [for details see Privalov et al. (1986)].

The transition of a protein or a single cooperative domain from the native to the denatured state is always accompanied by a significant increase of its partial heat capacity (see, for reviews, Sturtevant, 1977 Privalov, 1979). The denaturationaJ increment of heat capacity A JCP = C° Cp amounts to 25-50% of the partial heat capacity of the native protein and does not depend noticeably on the environmental conditions under which denaturation proceeds (Fig. 1) or on the method of denaturation. However, it is different foi different proteins and seems to correlate with the number of contacts between nonpolar groups in native proteins (Table I). On the other hand, the partial specific heat capacities of denatured states of different proteins appear to be rather similar (Tiktopulo et... 

Fig. 3. Denaturational increment of the partial specific heat capacity of pancreatic ribo-nuclease A (RNase), hen egg-white lysozyme (Lys), and sperm whale myoglobin (Mb). The dashed lines represent the parts of these functions that were obtained by a linear extrapolation of th partial heat capacity of the native state. The dot-and-dash lines show the behavior when the values measured at 50°C are assumed to be temperature independent.

The main consequence of a heat capacity difference between native and denatured states of a protein is that the thermodynamic functions that determine the transition between these states are all temperature-depen-dent. Indeed, since... 

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.

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 ... 

The denaturational increment of the heat capacity might be described partly by the increase of the extent of configurational freedom of the protein molecule upon denaturation. However, as was shown by Sturte-vant (J977) and Velicelebi and Sturtevant (1979), the contribution of this effect to the observed denaturational increment of the protein heat capacity cannot be large. This conclusion becomes especially evident from the impossibility of using this configurational effect alone to explain the negative values of the enthalpy and entropy of protein denaturation at low temperatures. 

The most plausible explanation for the significant denaturational increment of the protein heat capacity is that it is due to water that comes in contact with the protein nonpolar groups exposed upon denaturation... 

Comparison of the enthalpy of protein denaturation (Table I) with the enthalpy of solution of liquid hydrocarbons at Ts (Table II) shows also a great difference in their values the enthalpy of protein denaturation at Ts is about 6 kJ per mole of amino acid residues with an average molecular weight of 1 IS the enthalpy of solution of hydrocarbons of comparable size (ethylbenzene, Afw = 106) is almost five times larger at this temperature. For denaturation of solutions of proteins in water AnCp(25°C) is about 70 J K-1 per mole of amino acid residues, whereas A"Cp(250C) for ethylbenzene is 318 J K-1 mol-1. However, this difference in the enthalpy and heat capacity increment is quite understandable, as not all of the groups in a protein are nonpolar, not all are screened from water in the native state, and not all are in contact with water in the denatured state. 

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