Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.
AUTOR(ES)
Garel, J R
RESUMO
The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 25 degrees. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn.HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is 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. Previous work has shown that 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.HCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn.HCl at pH 5.8. We conclude that 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, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.
ACESSO AO ARTIGO
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