THE PHOTOINACTIVATION OF TRYPSIN AS SENSITIZED BY METHYLENE BLUE AND EOSIN Y*

Abstract— The kinetics of the photoinactivation of trypsin as sensitized by methylene blue and by eosin Y were investigated. The time-course of inactivation was first-order. The rates of inactivation were essentially identical as measured by three methods: the casein assay, the benzoyl-L-arginine ethyl ester assay, and the hemoglobin (in 5 M urea) assay. This is in sharp contrast to the results of earlier studies with flavins as photosensitizers, where the rate of inactivation was found to be much greater when measured by the hemoglobin assay than when measured by the other two assays. These differences are interpreted as indicating the production of a urea-sensitive 'damaged class' of trypsin molecules during photodynamic inactivation with flavins as sensitizers, but not with methylene blue and eosin Y. The results presented indicate that the mechanism of the photodynamic inactivation of enzymes might be different with different photosensitizing dyes.