title: Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.
abstract: Inactivation, dissociation, and unfolding of the homodimeric glutathione transferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibrium, using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence, far UV circular dichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M the enzyme progressively dissociates into structured monomers. At higher denaturant concentrations the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only by starting from the enzyme denatured at concentrations below 0.5 M. The enzyme denatured at GdmCl concentrations higher than 0.5 M only partially refolds. Globally our results indicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i.e., inactivation of the structured dimer, dissociation into partially structured monomers, followed by complete unfolding.
authors.affiliation: Dipartimento di Scienze Biomediche, Università "G. D'Annunzio", Via dei Vestini, Chieti, 66013, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Sacchetta
authors.forename: A
authors.initials: A
authors.lastname: Pennelli
authors.forename: T
authors.initials: T
authors.lastname: Bucciarelli
authors.forename: L
authors.initials: L
authors.lastname: Cornelio
authors.forename: F
authors.initials: F
authors.lastname: Amicarelli
authors.forename: M
authors.initials: M
authors.lastname: Miranda
authors.forename: C
authors.initials: C
authors.lastname: Di Ilio
mesh_terms: D000818:Animals; D002023:Bufo bufo; D002942:Circular Dichroism; D019281:Dimerization; D004625:Embryo, Nonmammalian; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects*; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D005982:Glutathione Transferase; D019791:Guanidine