Spectroscopic method for measuring activity of cis-aconitate decarboxylase, an important metabolic regulator of...

Cis-aconitate decarboxylase (ACOD1) is a key enzyme converting cis-aconitate to itaconate, which has therapeutic potential for inflammatory diseases. Existing methods to measure ACOD1 activity and itaconate are often expensive and complex. We developed a novel, high-throughput spectrophotometric assay using the Fürth-Herrmann reaction. Our method quantifies ACOD1-catalyzed itaconate production by leveraging distinct absorbance ratios of cis-aconitate and itaconate at 386 nm and 440 nm. We optimized parameters, characterized human ACOD1 kinetics, and determined an IC₅₀ for citraconate consistent with previous reports. This simple, fast, and reliable assay, requiring only a UV-Vis spectrophotometer, will accelerate screening for ACOD1 modulators, speeding up therapeutic development.