ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET‐INDUCED INACTIVATION OF β‐TRYPSIN*

Abstract

Abstract— Stern‐Volmer quenching constants for β‐trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern‐Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β‐trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β‐trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength. Investigations of the ultraviolet‐induced inactivation of β‐trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β‐trypsin is not involved in enzyme inactivation or tryptophan destruction.