Binding of serotonin and dopamine to 'serotonin binding proteins' in bovine frontal cortex: evidence for iron-induced oxidative mechanisms.

Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.