Assay for tyrosine hydroxylase in hypothalamic homogenates using high-performance liquid chromatography with electrochemical detection.

A highly sensitive and reliable assay for tyrosine hydroxylase (TH) activity in hypothalamic homogenates of male rats using high-performance liquid chromatography with electrochemical detection is described. Modification of sample preparation and chromatographic conditions led to a complete separation of L-3,4-dihydroxyphenylalanine (L-DOPA) and alpha-methyldopa from all interfering catecholamines and their metabolites. This assay is highly sensitive; 2 pmol of L-DOPA formed enzymatically could be measured. We were able to determine TH activity in tissue pieces weighing less than 1 mg. TH activity was not changed after storage for three months at -80 degrees C. In hypothalamic homogenates the Michaelis constant (KM) for L-tyrosine was 80.5 +/- 6.5 mumol/l and the maximal velocity (Vmax) was 132.5 +/- 10.5 pmol/mg of protein per min for L-DOPA formed enzymatically.