Conformation of NADH studied by fluorescence excitation transfer spectroscopy

Dihydronicotinamide adenine dinucleotide (NADH), is known to stack in two l imiting conformations. Surprisingly, previous experimental work on NADH has not clearly defined whether this folding and unfolding process can be desc ribed as first order (involving only two states) or whether one or more int ermediates must be included in the description. In addition, a large dispar ity exists between reported equilibrium constants for the aqueous solution at room temperature. Using methanol as a denaturant, we have used fluoresce nce excitation transfer spectroscopy to probe the stacking/un-stacking equi librium. Our results can be represented using a simple two state model. Fur thermore, the mole fraction of aqueous NADH in the stacked configuration is significantly higher than previously reported. Using a thermodynamic two s tate model, we have determined DeltaG degrees (H2O) = 8.8 +/- 1.4 kJ mol(-1 ). From excitation energy transfer measurements the fraction of NADH in the folded form at 295 K was determined to be 0.55. (C) 2001 Elsevier Science B.V. All rights reserved.