Bovine trypsin and α-chymotrypsin were inactivated by photodynamic treatment in sodium phosphate buffer at pH 8 using methylene blue, eosin Y and FMN as sensitizers. Measurements sensitive to changes in protein conformation, in particular, tryptophan fluorescence, rates of unfolding in 8 M urea, ion-exchange chromatography and thermodenaturation kinetics were made on the enzymes during the course of inactivation. The tertiary structures of trypsin and chymotrypsin are altered as measured by these methods but the rates of change in conformation do not parallel the rates of loss of enzyme activity during photodynamic treatment. Further, active enzyme molecules are produced by photodynamic treatment which are more easily denatured by high concentrations of urea than is native enzyme. This study demonstrates the necessity of ruling out effects on enzyme secondary and tertiary structure before interpreting photodynamic inactivation in terms of the photooxidation of critical amino acid residues in the active site region.

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