SOLVENT ISOTOPE EFFECTS ON AZURIN THERMAL UNFOLDING

The thermal unfolding of azurin in D2O has been investigated by differ ential scanning calorimetry, optical density measurements, and electro n paramagnetic resonance spectroscopy, The study has allowed us to rel ate the local conformational changes occurring around the active site of azurin with the unfolding of the whole protein as the temperature i ncreases. DSC and OD experiments have shown that the thermal unfolding is, on the whole, irreversible and kinetically controlled. Moreover, by extrapolation of both the experimental heat capacity and the optica l density data to infinite heating rate, we have separated the reversi ble step from the irreversible, kinetically controlled one and calcula ted the thermodynamic parameters of the time-independent part of the d enaturation process. The whole of the results suggest that the unfoldi ng of azurin in D2O follows the same pathway as observed in H2O (La Ro sa et al. J. Phys. Chem. 1995, 99, 14864-14870),(1) but it is shifted to a higher temperature. From the comparison of the thermodynamic unfo lding parameters obtained in the two solvents, it results that D2O des tabilizes the native state of the protein. According to an analysis of the thermodynamic behavior of model compounds in heavy water, this de stabilizing effect can be mainly ascribed to the apolar groups of the protein. In addition, the region around the active site is enthalpical ly less influenced by changing the solvent with respect to the global protein. This behavior has been ascribed to the different solvent-azur in interactions in heavy and in light water. Finally, EPR results show that during the thermal unfolding, the active-site geometry changes f rom trigonal bipyramidal to square planar. Such a conformational chang e is not influenced by solvent isotopic effects.