A simple and sensitive fluorescence assay for tyrosine hydroxylase activity

Abstract A simple and sensitive fluorescence assay for tyrosine hydroxylase (TH) activity wasdevised based on rapid isolation of enzymatically formed dopa by a double-column procedure fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminum oxide). Interfering substances were removed by the first Amberlite CG-50 column. Dopa was adsorbed on the second aluminum oxide column, then eluted with 0.5 m acetic acid, and assayed by the highly sensitive hydroxyindole method of Johnson et al. (1973, Anal. Biochem. 54 , 129–136). The standard incubation mixture (total volume, 0.5 ml) contained 0.3 m m l -tyrosine, 1.0 m m 6-methyl-5,6,7,8-tetrahydropterin, 100 m m mercaptoethanol, and an optimal concentration of ferrous ion. d -Tyrosine was used for the blank incubation. Recovery of dopa added to the standard incubation mixture as internal standard was about 70% and was reproducible. The fluorescence characteristics of the product were the same as those of authentic dopa. Blank fluorescence was very low even with crude enzyme preparations. The limit of sensitivity was 100 pmol of dopa formed, which is close to the sensitivity of radioassays. TH activity in homogenates of rat brain stem or human putamen could be assayed in the standard incubation system containing ferrous ion. The validity of this fluorescence assay has been shown by the agreement between the values obtained by this method and by radioassay using l -[ U - 14 C]tyrosine as substrate. In the rapid assay procedure dopa in the eluate from aluminum oxide was assayed directly by native fluorescence. Although the sensitivity was about 1 nmol, this rapid assay procedure was found to be particularly useful for the purification of TH.