The Interaction of Tetraiodofluorescein with Glutamate Dehydrogenase from Bovine Liver

Reports from various laboratories have dealt with the binding properties of glutamate dehydrogenase from bovine liver (EC 1.4.1.3) (Pantaloni & Dessen, 1969; Huang & Frieden, 1969; Malcolm, 1972; Brown et al., 1973; Koberstein & Sund, 1973), but unfortunately these reports differ considerably over the number and nature of glutamate dehydrogenase coenzyme-binding sites as well as the magnitude of their dissociation constants. Fluorescence spectroscopy (Pantaloni & Dessen, 1969; Huang & Frieden, 1969; Brown eta] . , 1973) and relaxation kinetics (Kempfle et al., 1974) indicated that there are two binding sites for NADH but only one for NADPH. To support these earlier findings difference-spectrophotometric studies were performed with tetraiodofluorescein (Erythrosin B) as indicator to study the binding properties of some nicotinamide nucleotides acting as coenzymes or effectors, since absorption difference spectrophotometric titrations with coenzymes as titrants had failed owing to the high absorbance of these compounds at higher concentrations. Since it is reported in the literature (Johnson, 1974; Wasserman & Lentz, 1971) that tetraiodofluorescein binds at the active site of nicotinamide nucleotide-dependent dehydrogenases, this dye appears to be an appropriate tool for studying nicotinamide nucleotide interactions with glutamate dehydrogenase. Further, the application of this compound has the great advantage that the perturbation of the dye absorption spectrum, indicating binding to the enzyme, falls in a wavelength range (between 500 and 600nm) far away from enzyme and nucleotide absorption.