Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.
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.
Sacchetta
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P
Dipartimento di Scienze Biomediche, Università "G. D'Annunzio", Via dei Vestini, Chieti, 66013, Italy.
Pennelli
A
A
Bucciarelli
T
T
Cornelio
L
L
Amicarelli
F
F
Miranda
M
M
Di Ilio
C
C
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
D007527:Isoenzymes; D005982:Glutathione Transferase; D019791:Guanidine