Intensity-dependent enzyme photosensitization using 532 nm nanosecond laser pulses

The intensity dependence of the rose bengal (RB)-photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 microM RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than approximately 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet-triplet absorption coefficient was determined to be 1.9 x 10(4) M-1 cm-1 at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico-mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.