The ''cold denaturation'' phenomenon is analyzed using an extension of the Planck-Benzinger thermal work function. For small molecules, reaction enthalpies are o o ลฝ . 298 o often obtained around room temperature, such that โฌH s โฌH T q H โฌC p dT, 298 o o and the heat of reaction is estimated in terms of the innate temperature-invariant ลฝ . o ลฝ . ลฝ enthalpy inherent chemical bond energy , โฌH T . Cottrell The Strength of Chemical o Bonds; Academic Press; New York, London, 1958; Chapter 3; pp. 21แ46; Chapter 4; pp. . o o ลฝ . 47แ70 pointed out 40 years ago that โฌH and โฌH T differ only by 1% in small 298 0 w ลฝ . x molecules, but in 1971 Benzinger Nature London 1971, 220, 100แ103 made the crucial observation that this difference is large in biological macromolecules due to the large ลฝ . magnitude of the heat capacity integrals thermal agitation energy . In the other words, w o o ลฝ .x for small molecules, โฌH y โฌH T is a correction of only a few percent, whereas for 298 0
biological macromolecules, the heat capacity integrals can be large, from 10% up to 50% of the total heat of reaction. In the case of T4 phage lysozyme, the thermal unfolding of w ลฝ . x wild type and mutant W138, W138Y, and 3W 128, 138, 158 3Y forms have heat capacity integrals that are some 10 times greater than the innate temperature-invariant enthalpy. In cases of protein unfolding such as the phage T4 phage lysozyme mutants, no thermodynamic molecular switch, unique to biological systems, is observed. It is apparent that use of the Planck-Benzinger thermal work function to evaluate the innate temperature-invariant enthalpy can be tremendously helpful in differentiating between native wild-type and closely-related mutant forms of protein. Therefore, this thermodynamic application should be essential to any future studies involving the site-directed, mutagenic approach to an examination of structure-function problems in proteins.