The thermodynamic properties and state diagram of the thermally induced aggregation of myoglobin with 1,4-dioxane were determined by DSC, circular dichroism (CD), dynamic light scattering (DLS), scanning electron microscopy (SEM), and density measurements. At a mole fraction (x) of 1,4-dioxane of around 0.10, myoglobin exhibited α-helical aggregation at 25 ºC, which transformed to spherical aggregation without thermal denaturation upon heating. The enthalpy change for the spherical aggregation was −565 kJ mol
−1 at x = 0.10, which was numerically greater than that for thermal denaturation of the native state, 428 kJ mol
−1, indicating that the aggregate conformation involved many intermolecular interactions. At x = 0.125 – 0.25, the α-helix partially transformed to a β-sheet at 25 ºC, which formed an amorphous aggregate upon heating. The thermal transition depends on the incubation time of the pre-transition state due to conformational changes in this state. The positive change in the partial specific volume of the aggregate indicates a large cavity volume and reduced hydration. On the other hand, the activation volume for the aggregation is negative, (−4.5 ± 1.3) × 10
2 cm
3 mol
−1, suggesting that the activated state has a structure with fewer cavities and/or is highly hydrated compared to the pre-transition state, probably due to partial unfolding.
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