Refolding kinetics of nitrated ribonuclease A

Abstract

Summary The refolding from concentrated Gu-HCl of nitrated RNase A is measured by the changes in absorbance of the three exposed nitrotyrosines. The slow refolding kinetics are complex, with a major faster phase and a minor slower phase (amplitude ratio: 80%–20%). The major phase resembles the main folding reaction of unmodified RNase A in the dependence on Gu-HCl of its rate and activation enthalpy. The minor phase has both a rate and activation enthalpy independent of Gu-HCl. These biphasic kinetics are observed in conditions where the folding reaction is probably rate-limited by proline cis - trans isomerization (in 2 M final Gu-HCl), suggesting that different prolines of the polypeptide chain are the origin of these complex kinetics.