Quantum Yield Studies of the Dye-Sensitized Photoinactivation of Trypsin

Abstract

In general, enzymes are not inactivated by long-wavelength ultraviolet radiation or by visible light, since most proteins do not absorb these radiations to any great extent. Many enzymes are rapidly inactivated, however, on illumination in the presence of appropriate sensitizing dyes and molecular oxygen; oxygen is consumed in the process. Phenomena of this type are usually termed by biologists. Shortly after the discovery of photodynamic action (1), it was shown that certain dyes accelerated the inactivation of enzyme preparations by sunlight (2). The earlier work on photodynamic action has been reviewed by Blum (3), Santamaria (4), and Spikes and Ghiron (5). More recently, the photodynamic inactivation of a number of crystalline enzymes has been examined (see ref. 6 for a listing). In this laboratory we have examined the kinetics of the photodynamic inactivation of the enzyme trypsin (5-7). This enzyme was selected for photodynamic studies because considerable work has been done on its inactivation by ultraviolet and ionizing radiation (see ref. 6), and its amino acid sequence is largely known (8). To compare the effectiveness of different dyes on the photodynamic inactivation of trypsin, it is necessary to make measurements of the quantum yield of the process. The present paper presents data on the effects of a number of reaction variables on the quantum yields for the photodynamic inactivation of trypsin as sensitized with methylene blue, eosin Y, and riboflavin-5'-phosphate (FMN). There appears to be only one report in the literature on quantum yields for the photodynamic inactivation of an enzyme; Shugar (9) found the quantum yield for the riboflavin-sensitized photoinactivation of lysozyme to be approximately 0.006.