Thermodynamic and kinetic properties of sorbitol-induced molten globule of myoglobin

To reveal the contribution of hydrophobic interactions to the stability of the molten globule (MG) state of proteins, the effect of sorbitol on the structure of acid-unfolded (AU) equine heart myoglobin was examined at pH 2 by means of circular dichroism (CD), stopped-flow CD, rheometry, and differential scanning calorimetry. The AU state of myoglobin was refolded by adding sorbitol to the MG state, which had a secondary structure and hydrodynamic volume similar to the native (N) state. The thermal denaturation of the MG state showed considerably small enthalpy change, low cooperativity, and small heat capacity compared to the N state unlike MG state of cytochrome c, indicating that the presence of the heme is important to preserve the strict tertiary structure of MG state not only N state, for heme proteins. The refolding was induced by preferential exclusion of three sorbitol molecules from the AU state compared to the MG state. The transition from the N-MG state kinetically proceeded via the AU state, followed by gradual refolding due to the preferential exclusion of sorbitol with 1.98 x 10(-3) s(-1) of kinetic constant at 3.3 M sorbitol and 10.9 A degrees C.