Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay

Tyrosine hydroxylase (TH) is the rate-limiting step in dopamine (DA) synthesis, oxidizing tyrosine to LDOPA, 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. (c) 2012 Elsevier Inc. All rights reserved.