The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5 '-dithiobis(2-nitrobenzoic acid) - A new method for the determination of ligand interactions with metallothionein

The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobeti zoic acid) (Nbs(2)) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At st oichiometries of Nbs(2) to MT from 0.5 to 5, the reaction followed first or der kinetics. The first order rate constants obtained were independent from the concentration of Nbs(2) but were linearly dependent on the concentrati on of MT. At higher Nbs(2)/MT stoichiometries, the reaction deviates from f irst order kinetics and the observed rate constant increases. The reactivit y of MT towards Nbs(2) has been probed at 4 muM concentration of both reage nts where the reaction is monophasic and is characterized by a linear Arrhe nius plot (E-a = 45.8 +/- 2.7 kJ.mol(-1)). It has been demonstrated that me tal release at low pH or subtraction from MT by EDTA substantially increase s the reactivity of MT towards Nbs(2). At the same time, a number of nonmet allic ligands moderately accelerate the reaction of NIT with Nbs(2) and hyp erbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constant s were calculated as follows: ATP, K = 0.31 +/- 0.06 mm; ADP, K = 0.26 +/- 0.07 mm. Several compounds such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations.