Quantum Yield Studies of the Dye-Sensitized Photoinactivation of Trypsin

Abstract

The quantum yield of the photodynamic inactivation of trypsin was studied with methylene blue, eosin Y, and FMN as sensitizers. The reaction was studied for each dye with respect to several kinetic variables: light intensity, dye concentration, trypsin concentration, pH and ionic strength, and temperature. Further, the effects of several protective agents, including sodium iodide, histidine, AET, and egg albumin, were examined. Quantum yields for inactivation under the reaction conditions used were 0.0014 with methylene blue, 0.0027 with eosin Y, and 0.0023 with FMN, and were essentially independent of light intensity. With increasing dye concentration, the quantum yields increased rapidly, attained a maximum, and then decreased; the yields increased with increasing enzyme concentration. No inactivation occurred below pH 6 with methylene blue and eosin Y. As pH was increased above this value, quantum yields rose sharply and then leveled off. With FMN, the same behavior was observed above pH 6, but, in contrast, appreciable inactivation occurred below pH 6. With all three dyes, the quantum yields increased slightly as the temperature was raised from 4[degree]C to 33[degree]C. Histidine and egg albumin protected trypsin against photodynamic inactivation with all three dyes; sodium iodide protected with eosin Y and FMN, and AET protected with methylene blue and FMN.