Acid-base equilibria of the oxidized beta-nicotinamide adenine dinucleotide-pyruvate adduct in the ground and electronically excited states. A proton transfer probe for proteins.
Proton transfer reactions of the oxidized NAD+-pyruvate adduct in its electronically excited singlet state were studied in aqueous solution and when bound to several proteins. The ionization constant of the adduct in the excited state was found to differ by several orders of magnitude from its value in the ground state. A rapid deprotonation reaction was found to follow electronic excitation if a suitable proton acceptor is present. The deprotonation is accompanied by a marked change in the fluorescence spectrum of the adduct and is therefore easy to detect and follow. The free adduct in aqueous solution was found to be in equilibrium between open and folded conformations of which only the open one is fluorescent. The equilibrium constant between these two forms was evaluated. A complex dependence of the fluorescence on the pH in the range of 1--7 was found, which originates in a shift of the equilibrium constant toward the open conformation and in protonation of acid groups of the adduct. Interaction of the adduct with four proteins was studied. Each had a different effect on the proton transfer reaction, reflecting differences in the microenvironment of the bound adduct. A possible use of the adduct as an excited-state proton transfer probe for proteins is presented.
D000429:Alcohol Oxidoreductases; D000818:Animals; D049673:History, 20th Century; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins; D011773:Pyruvates; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D011506:Proteins; D011773:Pyruvates; D009243:NAD; D012710:Serum Albumin, Bovine; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase