Fluorescence Properties of Reduced Thionicotinamide - Adenine Dinucleotide and of Its Complex with Octopine Dehydrogenase

Abstract

Reduced 3-thionicotinamide-adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 .+-. 0.5) .times. 10-4. The interaction between sNADH and [Pecten maximus] octopine dehydrogenase was investigated by UV difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine. There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40 nm blue shift of the emission maximum. The protein fluorescence is quenched almost completey. The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring in the free form. These features appear to be unique to the octopine .cntdot. sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. sNAD neither binds nor acts kinetically as a coenzyme. sNADH exhibits relatively good binding, with Km and Ki values close to those of the natural coenzyme, but the turnover number if 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH Kd were determined as a function of temperature, and appear to be practically temperature-independent in the range 10-40.degree. C. The interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed.