Enthalpy, protein stability

Protein catalyst stability is limited. This is one of major drawbacks of enzymes. They commonly require temperatures around ambient to perform (15-50°C), pH values around neutral (pH 5-9), and aqueous media. In addition, any number of system components or features such as salts, inhibitors, liquid-gas or liquid-solid interfaces, or mechanical stress can slow down or deactivate enzymes. Under almost any condition, native proteins, with their Gibbs free enthalpy of stability of just a few kilojoules per mole, are never far away from instability. In this book, we cover inhibitors (Chapter 5, Section 5.3) or impeding system parameters (Chapter 17) and successful attempts at broadening the choice of solvents (Chapter 12). 

ProTherm (16) is a large collection of thermodynamic data on protein stability, which has information on 1) protein sequence and stmcture (2) mutation details (wild-type and mutant amino acid hydrophobic to polar, charged to hydrophobic, aliphatic to aromatic, etc.), 3) thermodynamic data obtained from thermal and chemical denaturation experiments (free energy change, transition temperature, enthalpy change, heat capacity change, etc.), 4) experimental methods and conditions (pH, temperature, buffer and ions, measurement and method, etc.), 5) functionality (enzyme activity, binding constants, etc.), and 6) literature. 

In brief, a DSC instrument comprises two cells fixed in an adiabatic chamber. One cell contains the sample to be tested, the second cell contains a reference solution or an empty DSC pan. The adiabatic chamber is maintained under pressure to avoid the evaporation of the sample (Plum, 2009). A DSC-thermogram represents the plot of heat capacity difference ACp (between the sample and the reference) as a function of temperature. Thermodynamic parameters, such as T, AH and AS, could be determined by the DSC curve analysis. T is the temperature at which the concentration of denatured and native forms of the protein are equal. This specific temperature is also called the midpoint of the thermal transition. AH represents the enthalpy of thermal transition determined from the integration of the DSC curve. The entropy (AS) of the thermodynamic transition of the protein may be calculated from the integrated area under the curve of AC /T vs. T. The free energy (AG), which gives an indication of the protein stability, can also be determined at any temperature from the values of AH and AS (O Brien and Haq, 2004 Plum, 2009). Thermal and thermodynamic properties of proteins analyzed by DSC are greatly affected by the experimental conditions used, such as pH, salts, alcohols, and the presence of other food components (e.g., lipids, polysaccharides) (Grinberg et al, 2009). 

The thermodynamic stability of a protein in its native state is small and depends on the differences in entropy and enthalpy between the native state and the unfolded state. From the biological point of view it is important that this free energy difference is small because cells must be able to degrade proteins as well as synthesize them, and the functions of many proteins require structural flexibility. 

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