A radiochemical assay for beta-ureidopropionase using radiolabeled N-carbamyl-beta-alanine obtained via hydrolysis of [2-(14)C]5, 6-dihydrouracil

A radiochemical assay was developed to measure the activity of beta-ureidopropionase in human liver homogenates which is based on the detection of the reaction product (14)CO(2) by liquid scintillation counting. Radiolabeled N-carbamyl-beta-alanine was prepared within 15 min by a simple hydrolysis of [2-(14)C]5, 6-dihydrouracil under alkaline conditions at 37 degrees 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 beta-ureidopropionase obeyed Michaelis-Menten kinetics with an apparent Km for N-carbamyl-beta-alanine of 15.5 +/- 1.9 microM. 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 CO(2).