The effect of methanol and temperature on the kinetics of refolding of ribonuclease A

Unfolded ribonuclease A consists of 20% fast refolding (Uf) and 80% slow refolding material (Us). The latter consists of at least two different forms which refold at different rates. We have used absorbance and fluorescence spectrophotometry to compare the kinetics of refolding in aqueous and aqueous-methanol solutions. At 1 degree C and pH 3.0, the addition of increasing concentrations of methanol (to 50%, v/v) had negligible effect on the rates and amplitudes of the slow refolding Us states. The effect of temperature on the two slow phases of refolding was determined in 35 and 50% methanol. From Arrhenius plots the energies of activation were found to be in the vicinity of 20 kcal/mol for both processes. The results suggest that both slow phases correspond to proline isomerization, and that the presence of methanol does not significantly perturb the overall refolding process. It is possible that the faster of the slow refolding phases corresponds to the isomerization of a proline residue which is trans in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.