Tyrosine Hydroxylase Enzyme Kinetics

The activity of purified TH and truncated forms was assayed at 37 °C^{7 (link)}. The purified enzymes were kept on ice and diluted with 0.5% (w/v) bovine serum albumin in 20 mM Na-Hepes pH 7, 200 mM NaCl and centrifuged at 10,000 × g for 5 min at 4 °C. The standard assay mixture contained 20 mM Na-Hepes, pH 7, 0.1 mg/ml catalase, 10 µM FAS, 50 µM L-Tyr and 0–800 µM DA. TH was added to a final concentration of 1 µg/ml (17 nM subunit) and preincubated at 37 °C for 1 min. The reaction was started by adding 200 µM BH4 and 5 mM DTT and stopped by adding one volume of 1% (v/v) acetic acid in ethanol after 5 min for TH, THNΔ35, THNΔ43, and THS40p and after 2 min for THNΔ70. Protein was removed by precipitation at −20 °C for 90 min followed by centrifugation at 20,000 × g for 14 min at 4 °C. The amount of L-Dopa in the supernatant was determined using a 1200 series high performance liquid chromatography (HPLC) system (Agilent technologies). The chromatographic separation was obtained using an Agilent Zorbax 300-SCX column with 20 mM HAc pH 3.5, 2% (v/v) propanol as mobile phase at a flow rate of 3 ml/min. The fluorescence detector was set to λ_ex = 280 nm and λ_em = 314 nm.