Journal article
Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay
Analytical biochemistry, Vol.432(1), pp.11-15
01/01/2013
DOI: 10.1016/j.ab.2012.09.005
PMCID: PMC3579528
PMID: 23010244
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 475nm. 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.
Details
- Title: Subtitle
- Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay
- Creators
- Lydia M Vermeer - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USAColin A Higgins - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USADavid L Roman - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USAJonathan A Doorn - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
- Resource Type
- Journal article
- Publication Details
- Analytical biochemistry, Vol.432(1), pp.11-15
- DOI
- 10.1016/j.ab.2012.09.005
- PMID
- 23010244
- PMCID
- PMC3579528
- NLM abbreviation
- Anal Biochem
- ISSN
- 0003-2697
- eISSN
- 1096-0309
- Publisher
- Elsevier Inc
- Language
- English
- Date published
- 01/01/2013
- Academic Unit
- Pharmacy; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984065487402771
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