Coupled Enzyme Assay for Measuring Ornithine Decarboxylase Activity in Cell Lysates Using a Liquid-Stable CO₂...

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine biosynthesis that plays a critical role in cell proliferation and tumorigenesis. Reliable quantification of ODC activity is essential for mechanistic and therapeutic studies. Traditional assays often rely on radiolabeled substrates or discontinuous endpoint measurements. Here, we describe a non-radioactive, continuous spectrophotometric assay for measuring ODC activity in cell lysates using a commercially available liquid-stable CO₂ detection reagent. In this assay, CO₂ generated by ODC is captured as bicarbonate and utilized in a coupled enzymatic system containing phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH), leading to oxidation of thio-NADH. The decrease in absorbance at 405 nm due to thio-NADH oxidation is monitored in real time and is proportional to ODC activity. The protocol is performed in a 96-well plate format, requires minimal reagent preparation, and is suitable for medium- to high-throughput applications. Key features • Non-radioactive, continuous assay for measuring ODC activity. • Utilizes a commercially available liquid-stable CO₂ detection reagent, requiring minimal preparation and enabling improved reproducibility. • Real-time monitoring at 405 nm using a standard microplate reader. • Adaptable to a high-throughput 96-well format.