A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

• Published in: Analytical biochemistry 2013-11, Vol.442 (1), p.19-23
• Main Authors: Jones, Andrew J.Y., Hirst, Judy
• Format: Article
• Language: English
• Subjects: Animals; catalytic activity; Cattle; Coupled assay; electron transport chain; Enzyme Activation; Escherichia coli - enzymology; Fumarate hydratase; Fumarate Hydratase - metabolism; Fumarates - chemistry; Fumarates - metabolism; hydratases; Kinetics; malate dehydrogenase; Malate Dehydrogenase - metabolism; malates; metabolic diseases; mitochondria; Mitochondria, Heart - enzymology; Mitochondria, Heart - metabolism; mutation; NADP (coenzyme); neurodegenerative diseases; Oxaloacetate decarboxylating malic enzyme; oxidation; prokaryotic cells; pyruvic acid; Spectrophotometry; succinate dehydrogenase (quinone); Succinate Dehydrogenase - metabolism; Succinate:ubiquinone oxidoreductase; succinic acid; Succinic Acid - chemistry; Succinic Acid - metabolism; tricarboxylic acid cycle
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2013.07.018

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP+ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.