The molten globule (MG) state, a compact denatured state with a significantly native-like secondary structure but a largely flexible and disordered tertiary structure, has been proposed to be a major intermediate of protein folding. To explore another approach for characterizing the MG state, sodium dodecyl sulfate (SDS) induced formation of the MG state of horse cytochrome c at pH 2 was studied by circular dichroism, visible spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). These techniques confirmed that the addition of SDS to acid-unfolded state of cytochrome c induced the MG state. Although, the DSC thermal denaturation of cytochrome c was always calorimetrically irreversible, the MG state induced by SDS at low concentrations showed a reversible profile. The spectroscopic properties demonstrated that the hydrophobic tail of SDS utilized the hydrophobic contribution to stabilizing the heme conformation at MG state in cytochrome c. This would be the main reason of thermal profile reversibility of MG state in cytochrome c. The reversibility of DSC thermogram would allow its deconvolution and analysis of the energetic domains for this protein. (C) 2003 Elsevier B.V. All rights reserved.