RADIOENZYMATIC ASSAY FOR TRYPTOPHAN-HYDROXYLASE - [H-3] H2O RELEASE ASSESSED BY CHARCOAL ADSORPTION

The rate-limiting reaction in the biosynthesis of the neurotransmitter , serotonin, is catalyzed by the enzyme, tryptophan hydroxylase. Studi es on the characteristics of this enzyme have been hampered by its rel ative instability and paucity in the brain. We have modified a charcoa l adsorption radioenzymatic assay used for the measurement of tyrosine hydroxylase to assess rat brain tryptophan hydroxylase activity. This protocol is based on the principle that aromatic amino acid hydroxyla ses are mixed-function oxygenases and will utilize O2 and reduced pter in to convert tritiated amino acid substrate to product and tritiated H2O. All product and unreacted substrate are adsorbed by acidified cha rcoal. The [H-3]H2O is analyzed by liquid scintillation spectrometry a nd is indicative (stoichiometrically) of the amount of product formed and, thus, the activity of the enzyme. This assay has a high signal-to -noise ratio and is sensitive enough to determine enzymatic activity i n homogenates of individual raphe nuclei. In addition, its simplicity in design allows for the simultaneous testing of large numbers of samp les. The enzyme activity and kinetic determinations derived from this protocol agree with those of other investigators using more lengthy, i nvolved procedures.