Denaturation of human copper-zinc superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study

Abstract

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of an active and heat‐stable monomeric form of Cu,Zn superoxide dismutase from the periplasmic space of Escherichia coli

“…As previously described [22] the Xenopus laevis CuZnSOD was less resistant to the heat treatment than the bovine enzyme and both the eukaryotic enzymes showed a concentration-dependent inactivation rate, suggesting that denaturation of the enzymes at high temperatures occurred upon monomerization. This hypothesis is supported by previous fluorescence studies on human CuZnSOD demonstrating that monomerization of CuZnSODs is favoured by dilution [23] and that the unfolding intermediate is a monomer that displays a molten globule state [24]. Furthermore, dissociation of bovine CuZnSOD as a function of protein concentration has also been demonstrated using gel filtration chromatography [25].…”

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

“…The shortening of the slow rotational correlation time and its lower relative contribution to the anisotropy decay as a function of denaturant concentration reflect the increasing weight of segmental motions. The apparent convergence of the fractions of the slow and fast components observed at higher denaturant concentrations is a consequence of the fact that the more the two rotational correlation times approach each other, the more difficult it is to discriminate the two populations, which ultimately tend to merge (57).…”

Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation

“…Numerous examples exist of compact, monomeric intermediates that are significantly populated during the equilibrium denaturation of a dimer. Some examples include the p2 subunit of tryptophan synthetase (Zetina & Goldberg, 1980), X repressor (Pabo et al, 1979;Banik et al, 1992), aspartate amino transferase (Herold & Kirschner, 1990;Leistler et al, 1992), phosphoglucose isomerase (Blackburn & Noltmann, 1981), superoxide dismutase (Mei et al, 1992), and glutathione S-transferase (Aceto et al, 1992;Sacchetta et al, 1993). In general, the intermediates have been detected indirectly by noncoincidental denaturation curves measured by methods that are sensitive to distinct structural features.…”