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

Abstract

Unfolded ribonuclease A consists of 20% fast refolding (U,) 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°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 truns in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.

Comparison of Folding Rates of Homologous Prokaryotic and Eukaryotic Proteins

“…The enthalpies of activation for refolding after a 40-min denaturation, as calculated from Arrhenius plots (0, 15, 25, and 37°C), were found to be 9, 16, and 20 kcal mol Ϫ1 for the activation of E. coli AspAT, cytosolic AspAT, and mitochondrial AspAT, respectively. Activation enthalpies in the range of 15-20 kcal mol Ϫ1 are generally thought to reflect Xaa-Pro trans-cis isomerizations (19,(22)(23)(24). After a 40-s denaturation, the refolding rates (Fig.…”