Chapter 11 381 the increased apparent heat capacity arising from heat energy uptake in the endothermic unfolding transition. Once this transition is complete, the thermogram reverts to a “post-transition” baseline, reflecting the heat capacity of the now-unfolded protein in solution. The transferred energy usually has two parts: the activation energy barrier (kinetic), and the enthalpic part (total heat absorption or release). The kinetic (activation energy) barrier determines the temperature and time dependence of the denaturation process. The total enthalpic heat change is calorimetrically measured when the phase transition takes place. As the temperature is raised, it becomes thermodynamically favorable for the protein to denature. The final denatured state can be at a higher or lower total energy than the original state. The final state is at a lower energy than the initial state when coagulation, aggregation, and/or gelation of denatured proteins occur, which is a strongly exothermic process. Many experimental methods for estimating thermodynamic parameters associated with protein transitions are based on the assumption/approximation of two-state behavior for the system. The accuracy of the data thus obtained, and the validity of its interpretation, are critically dependent on the validity of this assumption. The simplest, but most widely used kinetic model to express the two-state behavior is the first order irreversible rate reaction model. For protein denaturation this assumes that the process of interest may be represented by a transition between two experimentally distinguishable states, native (N) and denatured (D): k N D where k is the rate constant. There is no significant population of intermediate states, and the transition may be brought about by changes in temperature, pH or denaturant concentration. The D state does not necessarily have to become a random coil, nor even fully unfolded during the two-state transition, and might continue to change, to become “more unfolded” as more denaturant is