A Convenient and Economical Spectrophotometric Assay for Ornithine Decarboxylase and Related Amino Acid...

Ornithine decarboxylase (ODC) catalyzes the conversion of l-ornithine into putrescine as the first and rate-limiting step in polyamine biosynthesis. Common assays used to measure ODC activity are often costly, involve expensive reagents and instruments, and require labor-intensive protocols. To address these issues, this research developed an economical and user-friendly spectrophotometric assay for ODC and other decarboxylases. The principle of the assay relies on the reaction of the ODC product, putrescine, with sodium 2,4-dinitrobenzenesulfonate (DNBS) under alkaline conditions, with heating at 70 °C for 1 h, generating a bis-2,4-dinitrophenyl derivative that is extracted in toluene and measured spectrophotometrically at 340 nm, providing a sensitive readout of enzyme activity. This assay demonstrates decent sensitivity, with a limit of detection (LOD) for inducible ODC activity as low as 0.21 μg/mL (5.1 pmol), and kinetic analysis revealed a K _m of 0.34 ± 0.01 mM and k _cat = 32 ± 0.33 s^-1. For the detection of putrescine, the product of ODC, the LOD was 4.8 nmol. Histamine detection by this method achieved an LOD of 18 nmol. This DNBS method was successfully applied to other decarboxylases, including bacterial histidine decarboxylase, d-ornithine/d-lysine decarboxylase (DOKDC) reaction with d-ornithine and a very slow substrate, d-arginine, and the very low activity C387A mutant DOKDC reaction with d-ornithine.