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

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.

• Publication year

  2013

• Venue

  Analytical Biochemistry

• Publication date

  2013-11-01

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.ab.2013.07.018PMID 23886887PMCID 3783901
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of fumarate hydratase activity.

  Confidence 0.91

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• The readout is stoichiometric, with a 1:1 relationship between succinate oxidation and NADPH formation, and avoids reliance on artificial electron acceptors.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• The assay is presented as a simple, quantitative, real-time method that is easy to implement for isolated enzyme samples, membrane preparations, and tissue homogenates.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• A coupled enzyme assay links fumarate production from succinate oxidation to NADPH readout through fumarate hydratase and oxaloacetate decarboxylating malic dehydrogenase.

  Confidence 0.98

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

• coupled enzyme assay

  assay, method

  A multi-enzyme assay format that converts the target reaction product into a spectrometrically detectable signal.

  Aliases: coupled assay

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• fumarate hydratase

  enzyme

  An enzyme that converts fumarate to malate as the first coupling step in the assay.

  Aliases: fumarase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• nadph spectrometric detection

  measurement method, readout

  The optical measurement of NADPH formation used as the assay signal.

  Aliases: NADPH readout, spectrophotometric readout

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• oxaloacetate decarboxylating malic dehydrogenase

  enzyme

  An enzyme used to convert malate to pyruvate while reducing NADP+ to NADPH in the assay.

  Aliases: malic dehydrogenase, MDH

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• succinate dehydrogenase

  enzyme, complex

  Respiratory complex II enzyme activity that oxidizes succinate to fumarate in the assay context.

  Aliases: complex II, succinate:ubiquinone oxidoreductase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

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