NADH-Dependent Oxidoreductase Activity Assay of OsAIM1 Using a Microplate Reader

Peroxisomal β-oxidation is a key step in jasmonic acid biosynthesis. Quantitative biochemical characterization of enzymes involved in the β-oxidation pathway is essential for validating their catalytic functions and comparing differences among genetic variants. Existing enzyme activity assays largely rely on chromatographic techniques to quantify substrate consumption or product formation, but these approaches are not well-suited for high-throughput or continuous kinetic measurements. Here, we describe a spectrophotometric assay based on a plate reader determining OsAIM1 enzymatic activity by monitoring the decrease in NADH absorbance at 340 nm. The method employs a 96-well plate reaction system, enabling real-time kinetic measurements and providing a standardized workflow for calculating reaction rates. Reaction components, protein concentration ranges, and data processing parameters were systematically optimized to ensure linearity, reproducibility, and quantitative accuracy. This assay is simple to perform, requires small reaction volumes, and offers relatively high throughput, making it suitable for functional characterization and kinetic analysis of NADH-dependent enzymes. Key features • Enzyme activity assay based on NADH absorbance changes, enabling real-time measurement. • Conducted in a 96-well plate format with a low reaction volume and minimal protein requirement. • Allows calculation of reaction rates and specific activity with good reproducibility. • Applicable to NADH-dependent enzymes and suitable for comparison between different samples.