THE THERMAL-DENATURATION OF STEM BROMELAIN IS CONSISTENT WITH AN IRREVERSIBLE 2-STATE MODEL

The thermal denaturation of bromelain, a cysteine proteinase from the papain family, was studied by means of circular dichroism (CD) and dif ferential scanning calorimetry (DSC). It was found that this process i s completely irreversible and apparently follows a simple two-state me chanism of the type N --> D. The activation energy, E, That characteri zes this reaction was calculated by the use of different approaches: ( i) the effect of heating rate on the temperature at which the transiti on is half completed; (ii) analysis of individual transition curves; ( iii) kinetic studies at fixed temperatures; and (iv) single DSC tracin gs. The obtained values for E were rather similar to one another, vary ing from 164 to 226 kJ/mol. In comparison, the total calorimetric enth alpy change was 334 kJ/mol. When a more complex mechanism is considere d (N reversible arrow U --> D), which takes into account the presence of a reversibly unfolded state (U), our results suggest that the rate- limiting step is precisely the formation of U. Calculation of the corr esponding activation enthalpy and entropy also seems to support this p roposal.