Fluence-rate-dependent photosensitized oxidation of NADH

The photosensitizing activity of dimethoxyhaematoporphyrin, excited by a laser pulse at 532 nm (YAG-Nd3+), was investigated using reduced nicotinamide adenine dinucleotide (NADH) as a substrate. The photo-oxidative modification of NADH was monitored by measuring the absorbance at 340 nm. The use of nanosecond pulses (15 and 0.5 ns) resulted in photosensitized NADH oxidation which depended on the fluence but not on the fluence rate up to a peak fluence rate of 10(7) W cm-2. At higher fluence rates a decrease in NADH photo-oxidation was observed, as well as on irradiation with picosecond pulses (35 ps). Stern-Volmer assay of the quenching by sodium azide revealed a decrease in quenching efficiency with increasing peak fluence rate. Oxidation of NADH was not suppressed by the addition of 20 mM sodium azide at peak fluence rates above 6 x 10(9) W cm-2. This observation, as well as the significant bleaching of dye absorption, indicates excitation of the photosensitizer into higher lying excited singlet states and the involvement of processes other than photodynamic action.