A Unique Molten Globule State Occurs during Unfolding of Cytochrome c by LiClO₄ Near Physiological pH and Temperature: Structural and Thermodynamic Characterization^†

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO₄ at pH 6.0 and 25 °C by observing changes in difference molar absorbance at 400 nm (Δε₄₀₀), mean residue ellipticities at 222 nm ([θ]₂₂₂) and difference mean residue ellipticity at 409 nm (Δ[θ]₄₀₉). The denaturation is a three-step process when measured by Δε₄₀₀ and Δ[θ]₄₀₉, and it is a two-step process when monitored by [θ]₂₂₂. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO₄-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl₂) suggests that LiClO₄ induces a unique MG state, i.e., (i) the core in the LiClO₄-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO₄-induced process, native state ↔ MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.