A simplified ¹⁴CO₂-trapping microassay for tyrosine hydroxylase activity

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.