| Abstract
| - The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity wasfollowed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively.The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined.The results show that two stable intermediates exist in the transition from the homodimeric native enzymeto the unfolded monomers: one (N2*) is a slightly more expanded, non-native, and active dimer, and theother is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used toreach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partialreactions are 4.5 (N2 ⇆ N2*), 65.8 (N2* ⇆ 2M), and 17.8 kJ/mol (M ⇆ U). It appears that TcTIMmonomers are more stable than those found for other TIM species (except yeast TIM), where monomerstability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensionalsimilarities, a remarkable heterogeneity exists in the unfolding pathways.
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