Time-resolved and steady-state fluorescence measurements of beta-nicotinamide adenine dinucleotide-alcohol...

beta-nicotinamide adenine dinucleotide (NADH)-alcohol dehydrogenase complex was compared to either UVA irradiation (364 nm; 50 mW cm-2; 0-60 min) or heat in order to investigate complex stability. Prior to irradiation, frequency-domain fluorescence lifetime measurements indicated the presence of two principal components having short (subnanosecond) and long (nanosecond) fluorescence lifetimes reflecting free and bound NADH respectively. UVA exposure resulted in decreased NADH fluorescence intensity concomitant with decreased absorption at 337 nm. However, UVA irradiation did not reduce the fractional contribution of the long-lived bound NADH. The photoinduced fluorescence decrease appeared to be caused by the formation of oxidized NAD+ and not on UVA-induced dissociation of the NADH-protein complex. Such dissociation, detected by a red-shifted fluorescence maximum and decreased fractional contribution of the nanosecond component, was observed when NADH-protein mixtures were heated.