Abstract
The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microheterogeneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 7224–7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state.
Highlights
Protein folding is biologically important for many reasons
Our results have shown that the time-resolved fluorescence decay of denatured human superoxide dismutase (HSOD) in 6.5 M guanidine hydrochloride (GdHCI) displays more heterogeneity than that of native HSOD [l]
A summary of the observations leading to this conclusion is in order: (1) at 3.5 GdHCl the steady-state anisotropy, (r >,of HSOD varies with protein concentration, (2) in contrast to the steady-state anistropy, the amide circular dichroism (CD) signal is constant over a wide range of HSOD concentration at all GdHCl concentrations, (3) the center of the lifetime distribution is relatively constant over the same range of HSOD concentration in 3.5 M GdHCl and (4) the width of the lifetime distribution increases as HSOD concentration decreases in 3.5 M GdHCl
Summary
Protein folding is biologically important for many reasons. Folding is necessary to initiate the function of various precursor enzymes [ll. Diates may be necessary for protein translocation across cell membranes [3]. Knowledge of protein folding could provide avenues toward designing novel enzymes ‘. Current research has established that some proteins exhibit an intermediate state during the folding/ unfolding process [4-71. Experimental methods confirming the existence of intermediates include circular dichroism (CD), nuclear magnetic resonance, infrared spectroscopy and intrinsic viscosity measurements [8-111. Another powerful method for investigating protein folding intermediates is through the fluo-
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