Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride

Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogena se I from Kluyverpmyces lactis (KIADH I) were investigated using guanidiniu m chloride (GdmCl) as denaturant. Protein transitions were monitored by enz yme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentration s between 0.3 and 0.5 M, the enzyme progressively dissociates into structur ed monomers through an irreversible reaction. At higher denaturant concentr ations, the monomers unfold completely. Refolding studies indicate that a t otal reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmC1 concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KIADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, follo wed by complete unfolding. (C) 2001 Elsevier Science B.V. All rights reserv ed.