The rate of equine liver alcohol dehydrogenase denaturation by urea. Dependence on temperature and denaturant concentration.

The kinetics of the irreversible urea denaturation of equine liver alcohol dehydrogenase have been studied as a function of temperature and urea concentration. The unfolding of the macromolecule, monitored by means of the phosphorescence properties of a deeply buried tryptophan residue, was found to be strictly a two-state process over the entire temperature range. It is characterized by a steep dependence on urea concentration typical of highly cooperative transitions and below room temperature it possesses large negative activation energies. The reaction is comparatively slow, does not seem to be preceded by a fast phase, and the rate-limiting step does not have the characteristics of proline isomerization. When the data are analyzed in terms of binding equilibria the temperature dependence results from an anomalously large change in heat capacity. Although this is a property of strong hydrophobic interactions in model compounds the slow rates of denaturation are best understood with a model of protein stability which emphasizes the cooperative nature of intramolecular interactions such as hydrogen bonding.