The study contributes to the development of experimental methods capable of detecting the intermediate states of proteins upon equilibrium denaturation. The urea-induced transition curves of bovine carbonic anhydrase II and bacterial luciferase obtained from steady-state and time-resolved fluorescence were compared. The dependence of the following fluorescence parameters on the urea concentration was analyzed: the shift of the steady-state emission spectrum, the lifetimes, and the spectral contribution of the lifetime components. It was found that for carbonic anhydrase the steady-state fluorescence does not reflect the multi-stage denaturation, in contrast to the time-resolved fluorescence parameters. This can be due to the fact that the intermediate states of carbonic anhydrase are formed at close urea concentrations. For bacterial luciferase the transition midpoints of unfolding stages are significantly separated by urea concentration scale, so they can be seen both in steady-state fluorescence spectra and in lifetimes change. The observed differences in the fluorescence parameters of two proteins were discussed in terms of tryptophan residues location and the general mechanisms of unfolding pathways.
intrinsic fluorescence of proteins, equilibrium denaturation, carbonic anhydrase II, bacterial luciferase, fluorescence lifetime, protein unfolding pathway
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