Extended theoretical analysis of irreversible protein thermal unfolding

The theoretical analysis of the protein denaturation model which includes an irreversible, exothermic and rate-limited step has been improved and applied to the DSC profile of Azurin. The two-step nature of the irreversible denaturation of globular proteins is usually depicted in the following simplified scheme: N <--> U <--> F, which is known as the Lumry and Eyring model. In most of the works concerning the thermal unfolding of proteins, it is usually assumed that the irreversible step of the process does not take place significantly during the short time the protein spends in the temperature range of the DSC transition, or if this is not the case, that this irreversible step occurs with a negligible thermal effect. As we will show, this last assumption cannot be accepted acritically; in fact we have found that in the case of Azurin an evident exothermic effect occurs at the end of the transition. In order to fit the experimental Cp(exc) profile of Azurin, we have analyzed a model in which the exothermic effects of the irreversible step and the variations of DeltaH with temperature are taken into account. Our model was first tested simulating a series of profiles and considering the effects of the variation of the parameters on the shape of the curves, and successfully used to fit the experimental calorimetric profile of Azurin.