Spectroscopic studies of stability of papain in aqueous organic solvents

The effects of different concentrations of the water-miscible organic solvents ethanol, 1,4-dioxane, tetrahydrofuran (THF) and acetonitrile (ACN) on the stability of papain in aqueous solution were studied. Papain exhibited high stability in aqueous ACN, ethanol and 1,4-dioxane. Decreases in the activity of the enzyme were observed at organic solvent concentrations above 60%. THF caused a dramatic reduction in activity even at low concentrations (5–10%). The solvent-induced structural changes were followed by means of circular dichroism (CD) and intrinsic fluorescence spectroscopy measurements. The decreases in enzyme activity at 90% THF or 90% 1,4-dioxane were accompanied by the loss of the tertiary structure. However, at 90% ethanol and 90% ACN, papain exhibited an increased amount of the α-helical conformation, with little change in the tertiary structure.