Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.

Abstract

The kinetics of the refolding reaction of RNase A from high concentrations of guanidine HCl [Gdn.cntdot.HCl] or urea are biphasic, and show 2 refolding reactions whose rates differ 450-fold at pH 5.8 and 25.degree.. Measurements of 2''-CMP binding during refolding, after stopped-flow dilution of Gdn.cntdot.HCl or urea, show that functional bovine pancreatic RNase A (EC 3.1.4.22) is formed in the fast and slow phases of the refolding process. The guanidine-unfolded state of RNase A is apparently an equilibrium mixture of fast- and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or urea-unfolded RNase A is the same as that in the heat-unfolded protein at pH 2. The fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated Gdn.cntdot.HCl solutions; the same proportion of the fast-refolding species is found from pH 2 to pH 6, and from 2 M to 6 M Gdn.cntdot.HCl at pH 5.8. The same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level. The structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.