Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae).
The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.
Abdalla
Abdel-Monem
AM
Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt. abdel_monem@hotmail.com
Hamed
Ragaa R
RR
D000818:Animals; D004795:Enzyme Stability / Q000502:physiology; D049851:Gastropoda / Q000201:enzymology*; D005982:Glutathione Transferase / Q000737:chemistry*; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea
D014508:Urea; D005982:Glutathione Transferase; D019791:Guanidine