The effect of methylurea, N,N′-dimethylurea,
ethylurea, and butylurea as well as guanidine hydrochloride
(GuHCl), urea and pH on the thermal stability, structural
properties, and preferential solvation changes accompanying
the thermal unfolding of ribonuclease A (RNase A) has been
investigated by differential scanning calorimetry (DSC),
UV, and circular dichroism (CD) spectroscopy. The results
show that the thermal stability of RNase A decreases with
increasing concentration of denaturants and the size of
the hydrophobic group substituted on the urea molecule.
From CD measurements in the near- and far-UV range, it
has been observed that the tertiary structure of RNase
A melts at about 3 °C lower temperature than its secondary
structure, which means that the hierarchy in structural
building blocks exists for RNase A even at conditions at
which according to DSC and UV measurements the RNase A
unfolding can be interpreted in terms of a two-state approximation.
The far-UV CD spectra also show that the final denatured
states of RNase A at high temperatures in the presence
of different denaturants including 4.5 M GuHCl are similar
to each other but different from the one obtained in 4.5
M GuHCl at 25 °C. The concentration dependence of the
preferential solvation change ΔΓ23, expressed
as the number of cosolvent molecules entering or leaving
the solvation shell of the protein upon denaturation and
calculated from DSC data, shows the same relative denaturation
efficiency of alkylureas as other methods.