This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 × 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 μg protein with 150 μM l-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37°C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 μg hog kidney aromatic-l-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37°C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37°C may reach 14,000 dpm, whereas the blank only has 300 dpm (< 3% of test value). This microassay is fast (< 2 h to complete all reactions) and convenient for performing a large number of determinations.
- tyrosine hydroxylase activity
ASJC Scopus subject areas