The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure‐induced unfolding

Abstract

We have studied the reaction native ⇆ denatured for the 33‐kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure‐induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (Δ V ). The change in Δ V was related to the sucrose and glycerol concentrations. The decrease in Δ V varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half‐unfolding pressure ( P 1/2 ) calculated from the net increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea‐ surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in Δ V showed a large contribution to the theoretical P 1/2 shift, suggesting that the Δ V change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the Δ V change is discussed. The rate of pressure‐induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers.