Evidence for an excited-state reaction contributing to NADH fluorescence.

The fluorescence of reduced β-nicotinamide adenine dinucleotide (NADH) was monitored as a function of the excitation and emission wavelengths. In aqueous and organic solvents the intensity decay was found to be more heterogeneous than reported earlier. When the ternary complex of NADH with the enzyme (horse liver alcohol dehydrogenase) and substrate analog (iso-butyramide) is formed, three exponents are required to fit the data. The decay-associated spectrum for the shortest lifetime undergoes a sign change from positive at the blue edge of emission to negative at the red edge. This phenomenon is interpreted as an outcome of reversible excited-state reaction leading to the appearance of at least one fluorescent product.