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

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 IC(50) 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.