The Photoinactivation of Trypsin as Sensitized by Methylene Blue and Eosin Y*

Abstract

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.

Quantum Yield Studies of the Photodynamic Inactivation of Trypsin

“…In contrast, Ghiren (12) and others (53) have more recently demonstrated the photodynamic inactivation of ribenuclease by riboflavin using other assay procedures.…”

“…in the presence of ascorbic acid than when free. In our trypsin system, it is not known whether the dye must be bound to the protein for photodynamic action'-to occur (12)(13). Pestemer (45) has measured the quantum yield of the anaerobig photochem ical reduction of thionine at 25' C in buffered fifty per cent ethanol.…”

“…Molecules as simple as phenol (8) and certain amino acids (9)(10)(11) can be altered by photodynamic treatment. More complicated molecules reported to be altered photodynamically include proteins (12)(13)(14)(15) and nucleic acids (16). Viruses can be modified or inactivated photodynamically (17)(18)(19).…”

“…Enzymes have.been used for many studies of this type since effects on catalytic activity can be measured. Photodynamic effects en many different enzymes, including 0(-amylase, carbonic anhydrase, chymotrypsin, cytochrome-£, dehydrogenases, hyaluronidase, lysozyme, myosin, pepsin, peroxidase, phosphoglucomutase, carboxypeptidase A, ribonuclease, urease, catalase, and enolase (12)(13)(14)(15), have been studied. All of the original work reported here has been carried out with trypsin.…”

“…Since that time, several modern reviews have been written that cover the subject thorough ly (14-15,2 2-26). Certain aspects of the field, especial ly effects on proteins , enzymes and amino acids, were reviewed extensiv ely in an earlier thesis from this laborato ry (12).…”