Synergistic inhibition of acid-induced protein denaturation by trehalose and NaCl: Thermodynamic and kinetic studies

• Trehalose and NaCl synergistically inhibit acid-induced protein denaturation.
• Trehalose and NaCl synergistically induce the formation of molten-globule state.
• Kinetic pathway changes greatly with the increment of the additives in the mixture.
• The rate constants for all the unfolding phases change slightly.
• Amplitudes for the kinetic phases diminish greatly with increasing the additives.

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.