The thermal denaturation of azurin in H2O, D2O and in ethanol-H2O mixtures has been investigated by electron spin resonance (ESR), optical absorption spectroscopy and differential scanning calorimetry (DSC). The OD625/T variation observed at a scan rate of 0.7°C/min in H2O shows a cooperative OD transition between 78 and 82°C. In this step the intense charge-transfer band of azurin at 625 nm disappears. The ESR spectra recorded at -153°C of the protein in the native state and after heating at 80 and 82°C indicate that both the symmetry and the copper ligands change with the thermal transition. The DSC measurements show that the thermal denaturation of azurin, which occurs at 84.4°C, is irreversible and kinetically controlled. This complex transition has been described as a multistep denaturation path and was analysed using a Lumry-Eyring type mechanism. The experimental C(p.exc) profile has been simulated and the calorimetric enthalpies related to the reversible and irreversible step, ΔH(u) and ΔH(ag), respectively, are obtained. The kinetically controlled steps have been investigated by means of optical and DSC measurements at different scan rates and the apparent activation energy, E(a), has been calculated. The denaturation of azurin in D2O and ethanol-H2O mixtures follows the same denaturation path as in H2O, although a shift of the OD625/T and DSC profiles is evidenced. The temperature of the thermal transition and the E(a) values decrease in ethanol-H2O mixtures, but increase in D2O.
|Titolo:||Experimental model for the thermal denaturation of azurin: A kinetic study|
|Data di pubblicazione:||1996|
|Appare nelle tipologie:||1.1 Articolo in rivista|