Equilibrium and kinetic studies of the counteraction of trehalose on acid-induced protein unfolding

It has been known that trehalose can counteract acid-induced protein denaturation and induce the formation of molten globule (MG) state, but the equilibrium and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on the acid-induced unfolding of ferricytochrome c was studied at pH 2.0. Far-UV circular dichroism, Soret absorption and fluorescence spectra indicate that trehalose inhibits acid-induced protein denaturation and induces the formation of MG state. The kinetics of the counteracting effects of trehalose was investigated by stopped-flow fluorescence spectroscopy. It is found that the unfolding of ferricytochrome c displays three phases (i.e., fast, intermediate and slow phases) in the absence of trehalose. At trehalose <0.8 M, the unfolding transition still occurs in three phases, but at trehalose >0.8 M, it is transformed into a biphasic process without the fast phase. The rate constants for all the unfolding phases decrease linearly with increasing trehalose concentration. Meanwhile, the increase in the fluorescence intensity for the fast and intermediate phases diminishes with the increment of trehalose concentration. Nevertheless, there is little influence of trehalose on the fluorescence intensity change in the slow phase. The studies have provided new insight into the effects of trehalose on the stability of proteins.

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► Trehalose inhibits the acid-induced denaturation of ferricytochrome c at pH 2.0.
► Trehalose induces the formation of molten globule state at pH 2.0.
► At trehalose concentrations <0.8 M, the unfolding transition occurs in three phases.
► At trehalose concentrations >0.8 M, the unfolding transition occurs in two phases.
► The unfolding rates decrease linearly with increasing trehalose concentration.