Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.
A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.
D000818:Animals; D001933:Brain Stem / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004295:Dihydroxyphenylalanine / Q000032:analysis; D006801:Humans; D008750:Methyldopa / Q000032:analysis; D011699:Putamen / Q000032:analysis; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis*
D008750:Methyldopa; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase