The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W5)I, clusters (W4)II and free water molecules WIII. The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(−ξwWI→ξwWII+ξwWIII+cavity) takes place, with expulsion from the bulk of ξw water molecules WIII, whereas in the processes of Class B the opposite transformation B(−ξwWIII−ξwWII→ξwWI−cavity) takes place, with condensation into the bulk of ξw water molecules WIII. The thermal equivalent dilution (TED) principle is exploited to determine the number ξw. The denaturation (unfolding) process belongs to Class A whereas folding (or renaturation) belongs to Class B. The enthalpy ΔHden and entropy ΔSden functions can be disaggregated in thermal and motive components, ΔHden=ΔHtherm+ΔHmot, and ΔSden=ΔStherm+ΔSmot, respectively. The terms ΔHtherm and ΔStherm are related to phase change of water molecules WIII, and give no contribution to free energy (ΔGtherm=0). The motive functions refer to the process of cavity formation (Class A) or cavity reduction (Class B), respectively and are the only contributors to free energy ΔGmot. The folded native protein is thermodynamically favoured (ΔGfold≡ΔGmot<0) because of the outstanding contribution of the positive entropy term for cavity reduction, ΔSred≫0. The native protein can be brought to a stable denatured state (ΔGden≡ΔGmot<0) by coupled reactions. Processes of protonation coupled to denaturation have been identified. In thermal denaturation by calorimetry, however, is the heat gradually supplied to the system that yields a change of phase of water WIII, with creation of cavity and negative entropy production, ΔSfor≪0. The negative entropy change reduces and at last neutralises the positive entropy of folding. In molecular terms, this means the gradual disruption by cavity formation of the entropy-driven hydrophobic bonds that had been keeping the chains folded in the native protein. The action of the chemical denaturants issimilar to that of heat, by modulating the equilibrium betweenWI, WII, andWIII toward cavity formation and negative entropy production. The salting-in effect produced by denaturants has been recognised as a hydrophobic hydration process belonging to Class A with cavity formation, whereas the salting-out effect produced by stabilisers belongs to Class B with cavity reduction. Some algorithms of denaturation thermodynamics are presented in the Appendices.
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