The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lip
ase (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 denat
uration, 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 whi
ch can be refolded into the native state, and D-F the final denatured-state
that can not be renatured. Ethanol (10%) increased the denaturation veloci
ty by decreasing the refolding step, D-1+Ca2+-->N Ca2+, which would be caus
ed by the stabilization of D-1 by ethanol.