Microplate Assays for Spectrophotometric Measurement of Mitochondrial Enzyme Activity
Spectrophotometric analysis of metabolic enzyme activity from homogenized tissues is a valuable method for investigating mitochondrial content and capacity. Enzyme activity is normally measured in single cuvette spectrophotometers, requiring a large sampl
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roduction Spectrophotometric assessment of mitochondrial enzyme activity is a simple, useful means to assess overall mitochondrial content and/or health and is used in both clinical diagnostics and research applications. Traditionally, these assays were performed in single cuvettes, limiting high-throughput capabilities. We have adapted these assays for higher-throughput measures in 96-well microplates. While traditional oxygen consumption measures in isolated mitochondria are considered a gold standard for assessment of mitochondrial function, advantages of microplate enzyme activity assays are their high-throughput nature and that enzyme activity can be measured in small quantities of previously frozen tissue or cell lysates. Likewise, though mass spectrometry metabolomic measures provide excellent precision and breadth of metabolite content, interpretation of these data will often benefit from assessment of directional changes in metabolism, which can be provided by assessment of mitochondrial enzyme activity. However, because these assays are performed in vitro with an abundance of substrate present in a lysed sample, they are considered an index of maximal Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_22, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Rachel C. Janssen and Kristen E. Boyle
enzymatic capacity and should not be interpreted as physiological activation. Over the past 75 years, many respected laboratories have developed and modified assays for each of the mitochondrial enzymes described here, using varied technologies such as radiochemical or spectrophotometric assessment. Here, we have adapted single cuvette measures of mitochondrial enzyme activity for use in spectrophotometer microplate readers. The citrate synthase assay is adapted from Frazier and Thorburn [1] and is often used as an index of mitochondrial content. Typically, activity of the other mitochondrial enzymes is referenced to citrate synthase activity to account for differences in mitochondrial content between samples. β-hydroxyacyl CoA dehydrogenase (β-HAD) assay is adapted from Lynen and Wieland [2] for estimating activity of β-oxidation. The iron-sulfur cluster of aconitase renders it sensitive to superoxide damage. Thus, diminished activity can be used as an index of oxidative stress. This microplate assay is adapted from Gardner et al. [3]. Singly, or in combination, mitochondrial ETS complex activity assays are useful for estimating mitochondrial function. Complex I and II assays are adapted from Bindoff et al. [4], while complex III and IV assays are adapted from Spinazzi et al. [5]. Together, these assays can give a general assessment of mitochondrial content, health, and activity. Each laboratory using these assays should adapt a preferred method by optimizing sample preparation and assay conditions to achieve a linear reaction with a high signal-to- background ratio.
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