A sensitive fluorometric assay for dopamine-beta-hydroxylase activity by high-performance liquid chromatography.
A sensitive and specific fluorometric assay for dopamine-beta-hydroxylase (DBH) activity is described. The main natural substrate, dopamine (DA), was used and incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by an aluminum oxide column and was analyzed by high-performance liquid chromatography (HPLC) with trihydroxyindole fluorescence. Epinephrine (EN) was added to the incubation mixture as an internal standard after incubation, and this assay was therefore highly reproducible. HPLC conditions were settled to elute the product, NE, prior to the substrate, DA, and the internal standard, EN, between NE and DA. Only catecholamines produced significant peaks, and therefore, this assay is highly specific. We applied this method to measure the DBH activity in human serum and cerebrospinal fluid.
D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000032:analysis; D004299:Dopamine beta-Hydroxylase / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D004837:Epinephrine / Q000032:analysis; D006801:Humans; D009638:Norepinephrine / Q000032:analysis; D013050:Spectrometry, Fluorescence
D004299:Dopamine beta-Hydroxylase; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine