Spectroscopic Studies on Papain and Some Inactive Derivatives

The fluorescence emission spectrum of papain is attributable to the tryptophan residues in this protein with little contribution from tyrosine residues. The pH dependence of the fluorescence intensity of unactivated papain follows closely the titration curve for a residue of apparent pK' of 6.6 at 25°, while that of active papain can be fitted by a curve obtained by the summation of the titration curves for groups of apparent pK' of 6.6 and 8.5 at 25°. Examination of the temperature dependence of the pH fluorescence profiles, as well as a study of several inactive papain derivatives in which the thiol group at the active site has been alkylated, indicates that the above pK' values can be ascribed to the side chain of a histidine residue and to the thiol group at the active site of the enzyme, respectively. The fluorescence of both active and unactivated papain is unchanged in the presence of ethylene glycol (40%, v/v), methanol (50%, v/v), or dioxane (20%, v/v). The circular dichroism spectra, as well as ultraviolet difference spectra produced by solvent perturbation, of active and unactivated papain were superimposable, indicating that activation of this enzyme is associated with little, if any, conformational change.