Regular ArticleA Radiochemical Assay for β-Ureidopropionase Using Radiolabeled N-Carbamyl-β-alanine Obtained via Hydrolysis of [2-14C]5,6-Dihydrouracil

Add time:08/15/2019    Source:sciencedirect.com

A radiochemical assay was developed to measure the activity of β-ureidopropionase in human liver homogenates which is based on the detection of the reaction product 14CO2 by liquid scintillation counting. Radiolabeled N-carbamyl-β-alanine was prepared within 15 min by a simple hydrolysis of [2-14C]5,6-dihydrouracil under alkaline conditions at 37°C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human β-ureidopropionase obeyed Michaelis–Menten kinetics with an apparent Km for N-carbamyl-β-alanine of 15.5 ± 1.9 μM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO2.

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