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 d...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1999-09, Vol.369 (1), p.100-106
Main Authors: Sacchetta, Paolo, Pennelli, Alfonso, Bucciarelli, Tonino, Cornelio, Lucia, Amicarelli, Fernanda, Miranda, Michele, Di Ilio, Carmine
Format: Article
Language:English
Subjects:
amphibian
Animals
Bufo bufo
Circular Dichroism
denaturation
Dimerization
Embryo, Nonmammalian
glutathione transferase
Glutathione Transferase - chemistry
Glutathione Transferase - metabolism
Guanidine - pharmacology
Isoenzymes - chemistry
Isoenzymes - metabolism
Kinetics
Protein Denaturation
Protein Folding
Protein Structure, Secondary - drug effects
Spectrometry, Fluorescence
unfolding
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Summary: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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1999.1324