# High-resolution native electrophoresis in-gel activity assay reveals biological insights of medium-chain fatty acyl-CoA dehydrogenase deficiency

## TL;DR

A new gel-based assay helps study how genetic variants affect the structure and function of a key fatty acid metabolism enzyme.

## Contribution

A novel native electrophoresis-based activity assay was developed to distinguish MCAD tetramers from other protein forms.

## Key findings

- The assay showed a linear correlation between MCAD protein amount and enzymatic activity for octanoyl-CoA.
- Clinically relevant MCAD variants revealed differences in protein shape, activity, and FAD content.
- The method can be applied to study other acyl-CoA dehydrogenase family members linked to metabolic disorders.

## Abstract

Medium-chain specific acyl-CoA dehydrogenase (MCAD) is a mitochondrial homotetrameric flavoprotein that catalyzes the first step in fatty acid beta-oxidation. MCAD deficiency arises from variants that either impair enzymatic activity or destabilize interactions between subunits, leading to protein aggregation. Standard enzymatic assays measure the overall MCAD activity but cannot differentiate between tetramers and other protein forms—critical for understanding the impact of pathogenic variants on structure destabilization. In this study, we adapted a native gel colorimetric assay to quantify the activity of MCAD tetramers separately from other protein forms, providing novel insights into how pathogenic variants affect MCAD structure and function. The assay showed a linear correlation between protein amount and enzymatic activity for octanoyl-CoA, a physiological MCAD substrate. Applying this method to clinically relevant MCAD variants allowed us to distinguish subtle differences in protein shape, enzymatic activity, and FAD content, offering profound implications for understanding the molecular basis of MCADD. This methodology can be extended to analyze variants in other acyl-CoA dehydrogenase family members—such as glutaryl-CoA, isovaleryl-CoA or short-chain fatty acyl-CoA dehydrogenases—that are implicated in disorders of fatty acid and amino acid metabolism.

The online version contains supplementary material available at 10.1038/s41598-025-24684-3.