| Description |
When enzymes are illuminated with visible light in the presence of and appropriate sensitizing dye and oxygen, a loss of enzymatic activity is observed. This phenomenon is called photodynamic action or inactivation. Oxygen is consumed in the process. The present work, quantum yield determinations were made on the photodynamic inactivation of the enzyme trypsin using methylene blue (MeB), eosin Y, and flavin mononucleotide (FMN) as the sensitizing dyes. Illumination was provide by a 500-watt projector or a high-pressure mercury arc equipped with multilayer interference filters such that transmission peaks appropriate to each dye were obtained. Light energy absorption was measured with a vacuum thermocouple-galvanometer combination which was calibrated with a standard lamp. Aliquots of the illuminated trypsin plus dye solution were taken at intervals and the tryptic activity remaining was assayed spectrophotometrically using the benzoyl-L-arginine ethyl easter (BAEE) assay. In stirred systems, the time-course of the photodynamic inactivation of trypsin was first-order within experimental error under all conditions examined. In unstirred systems the results with Meb and eosin Y were the same as in stirred systems. With FMN as sensitizer, however, the rate of inactivation in unstirred systems was very low, and the reaction deviated markedly from a first-order process. On the basis of other studies in this laboratory, these latter results could be interpreted as demonstrating a difference dependence on oxygen partial pressure for the FMN-sensitized system. The effect of dye concentration on the quantum yield of trypsin photo inactivation was studied. MeB-sensitized systems have a quantum yield maximum of 0.0014 at 12.5 uM, eosin Y-sensitized systems have a quantum yield maximum of 0.0027 at uM, and FMN-sensitized systems have a maximum value of 0.0023 at 150 uM. These data indicate that the three mechanisms of actions are probably different. The quantum yield is independent of light energy absorbed over the range studied (0 - 0.01 watts/cm2) for all three dye-sensitized systems. The quantum yield decreases with increasing trypsin concentration over the range of 5 - 42 uM trypsin for all three dye-sensitized systems. These results are difficult to interpret at this time. The quantum yield to trypsin photo inactivation was examined over the pH range 2 - 10. No inactivation is evident for MeB- or eosin Y-sensitized systems at pH values below about 6. FMN-sensitized systems show low but significant quantum yield values from pH 2 - 6. All three dye-sensitized systems show high quantum yield values above pH 7. FMN may operate by different mechanisms on the acid side and basic side of the pH scale. These results can be interpreted to mean that the enzyme is folded in such a way that it cannot be inactivated readily in acid. The quantum yield in all three dye-sensitized enzymes has been shown to be unaffected by changes in the concentration of sodium phosphate buffer present over the range 0.00125 M - 0.125 M when the pH is held constant. Similarly, sodium chloride concentration up to 1.0 M has no effect on the eosin Y and FMN systems. Meb is precipitated out of high salt concentrations. The quantum yield of trypsin photo inactivation is an increasing linear function of temperature for all three dye-sensitized systems. Temperature studies give experimental activation energies, Ea, of from 2 - 5 kcal/mole for the photodynamic inactivation of trypsin. These values indicate either rate limiting thermo chemistry of some sort or the involvement of diffusion-controlled processed that require little energy. Many compounds are known to inhibit photodynamic action; these act by various mechanisms. Sodium iodide was found to inhibit trypsin photo inactivation in FMN- and eosin Y-sensitized systems, presumably by quenching the long-lived exited (triplet) state of the dye; it causes precipitation of Meb in manner analogous to sodium chloride, The MeB- and eosin Y-sensitized systems are inhibited by molar mounts of sodium iodide, while the FMN system is inhibited strongly by only micro molar amounts. These data also show the differences in the mechanisms between the FMN-sensitized system and the other two systems. Histidine, S-(2aminoethyl) isothiuronium bromide hydro bromide (AET), and egg albumin were used as inhibitors; they presumably act as inhibitors because they can be competitively photo oxidized. Histidine and egg albumin cause inhibition in all three dye-sensitized systems. AET inhibits Meb-and FMN-sensitized systems; it causes precipitation of eosin Y. All of these data are useful in evaluating possible reaction schemes for photodynamic action because any mechanism proposed must fit these kinetic parameter limitations. No currently proposed mechanism fits our experimental observations exactly, and further, these data support the conclusion that the mechanisms of action are different, depending on the dye used as the sensitizer. Additional types of studies will be needed to determine the exact reaction mechanism |
