Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation.

Abstract The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1–10. Equilibrium unfolding with urea showed that values of D 1/2 (5.7 M) and Δ G H 2 O (12.8 kcal/mol) were highest at pH 5.0, its p I and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of Δ G H 2 O although GdnHCl was 2-fold more potent denaturant with D 1/2 value of 3.1 M. The curves of fraction unfolded ( f U ) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0–3.0 and 6.0–9.0. Thermal denaturation in the pH range 4.0–5.5 resulted in aggregation and precipitation of the protein above 55 °C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission λ max and enhancement of fluorescence intensity of ANS at 70 °C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with α-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge–charge repulsion at the p I of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F⇄U mechanism. The pH dependence of stability described by Δ G H 2 O (urea) and Δ G (25 °C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.