title: Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay.
abstract: Tyrosine hydroxylase (TH) is the rate-limiting step in dopamine (DA) synthesis, oxidizing tyrosine to l-DOPA, which is further metabolized to DA. Current assays for monitoring activity of this enzyme require extensive work-up, require long analysis time, and measure end points, thereby lacking real-time kinetics. This work presents the development of the first real-time colorimetric assay for determining the activity of TH using a plate reader. The production of l-DOPA is followed using sodium periodate to oxidize l-DOPA to the chromophore dopachrome, which can be monitored at 475 nm. Advantages to this method include decreased sample analysis time, shorter assay work-up, and the ability to run a large number of samples at one time. Furthermore, the assay was adapted for high-throughput screening and demonstrated an excellent Z-factor (> 0.8), indicating suitability of this assay for high-throughput analysis. Overall, this novel assay reduces analysis time, increases sample number, and allows for the study of activity using real-time kinetics.
authors.affiliation: Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Vermeer
authors.forename: Colin A
authors.initials: CA
authors.lastname: Higgins
authors.forename: David L
authors.initials: DL
authors.lastname: Roman
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Doorn
mesh_terms: D002863:Chromogenic Compounds / Q000737:chemistry; D004298:Dopamine / Q000032:analysis / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D010504:Periodic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics; D013053:Spectrophotometry; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002863:Chromogenic Compounds; D011994:Recombinant Proteins; D010504:Periodic Acid; C009288:metaperiodate; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine