Two-phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol

The dynamics of the unfolding process of bovine pancreatic ribonuclease A ( RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 we re investigated in alkaline phosphate-buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance (H-1 NMR) spectra. Three N MR spectral parameters including Shannon entropy, mutual information, and c orrelation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours , and the kinetics of the unfolding pathway described by the three paramete rs is best fit by a model of two consecutive first-order reactions. Tempera ture greatly influences the rate constants of the unfolding kinetics with d ifferent temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest t hat the unfolding process of RNase A induced by low concentrations of DTT i s a two-phase pathway containing fast and slow first-order reactions.