Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chlorid e (GdmC1) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmC1, less than 1.0 M, the fluore scence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concent rations of denaturant (between 1.2 and 1.6 M GdmC1). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilin o-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Fur ther changes in the fluorescence properties occurred at higher concentratio ns of GdmC1, more than 1.6 M, with a decrease in emission intensity and a s ignificant red shift of the emission maximum from 340 to 354 nm. In this st age, the secondary structure was completely broken. A study of ape-enzyme ( Zn2+-free enzyme) produced similar results. However, comparison of the chan ges of the fluorescence emission spectra of native (Holo-) enzyme with Zn2-free (Apo-) enzyme at low GdmC1 concentrations showed that the structure o f the Hole-enzyme was more stable than that of the Ape-enzyme. (C) 1999 Els evier Science B.V. All rights reserved.