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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\u2014critical 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\u2014such as glutaryl-CoA, isovaleryl-CoA or short-chain fatty acyl-CoA dehydrogenases\u2014that are implicated in disorders of fatty acid and amino acid metabolism.<\/jats:p>","DOI":"10.1038\/s41598-025-24684-3","type":"journal-article","created":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T14:30:38Z","timestamp":1761229838000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["High-resolution native electrophoresis in-gel activity assay reveals biological insights of medium-chain fatty acyl-CoA dehydrogenase deficiency"],"prefix":"10.1038","volume":"15","author":[{"given":"Sergio","family":"Guerrero-Castillo","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alice","family":"Gr\u00fcn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole","family":"Lewandowski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Polina","family":"Gundorova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa Ela","family":"Blettenberger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nora Constanze","family":"Laubach","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katrin","family":"K\u00fcchler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Madalena","family":"Barroso","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlotte","family":"Uetrecht","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S\u00f8ren W.","family":"Gersting","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,10,23]]},"reference":[{"key":"24684_CR1","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1146\/annurev-physiol-021115-105045","volume":"78","author":"SM Houten","year":"2016","unstructured":"Houten, S. 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