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. 2025 Jul 11:706:115944.
doi: 10.1016/j.ab.2025.115944. Online ahead of print.

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

Kevin Knowlan  1 , Cody L Hoop  2 , Nadya I Tarasova  3
Affiliations

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

Kevin Knowlan et al. Anal Biochem. .

Abstract

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.

Keywords: Immune-Responsive Gene 1 (IRG1); Itaconate; Spectrophotometry; cis-aconitate decarboxylase (ACOD1).

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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