Necessity of ascorbic acid in the radioligand binding assay for [ 3H]5-hydroxytryptamine

Evidence is presented to suggest that ascorbic add is required in the radioligand binding assay for [ 3H]5-hydroxytryptamine ([ 3H]5-HT, [ 3H]serotonin). In the absence of ascorbic acid, oxidation of [ 3H]5-HT occurred if the radioligand solution was left on ice for 1 or 3 hr. The oxidative products were detectable by thin-layer chromatography. They increased the binding significantly, although there was only slight oxidation (< 1%) of the [ 3H]5-HT. When ascorbic add was not used in the radioligand binding assay for [ 3H]5-HT, even though [ 3H]5-HT was prepared immediately before incubating with the membranes from the cortex of the rat, the binding also increased. The increased binding of [ 3H]5-HT had a low affinity (k d = 14 nM) and high B max (1180 fmol/mg protein), compared to that in the presence of ascorbic add (K d = 5 nM; B max = 210 fmol/mg protein). However, the increased binding was not receptor-related because the additional radioactivity was not displaceable by excess of unlabelled 5-HT (10μM) or d-LSD ( d-lysergic add iethylamide) and anomalous “spedfic binding” occurred in boiled membranes from cortex, in which the binding sites for 5-HT were destroyed. These results suggest that oxidation of [ 3H]5-HT may occur during the incubation with membranes and that ascorbic add is therefore required as an antioxidant.