Guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase

The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation, The denaturation process was well explained consistently by a two-step mechanism, as follows: [GRAPHICS] where N is the native state of PCL, D-1 an intermediate denatured-state which can be refolded into the native state, and D-F the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D-1+Ca2+-->N Ca2+, which would be caused by the stabilization of D-1 by ethanol.