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

Anal Biochem. 2013 Nov 1;442(1):19-23. doi: 10.1016/j.ab.2013.07.018. Epub 2013 Jul 22.

Abstract

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.

Keywords: Coupled assay; Fumarate hydratase; Oxaloacetate decarboxylating malic enzyme; Succinate:ubiquinone oxidoreductase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Enzyme Activation
  • Escherichia coli / enzymology
  • Fumarate Hydratase / metabolism
  • Fumarates / chemistry
  • Fumarates / metabolism
  • Kinetics
  • Malate Dehydrogenase / metabolism
  • Mitochondria, Heart / enzymology
  • Mitochondria, Heart / metabolism
  • Spectrophotometry
  • Succinate Dehydrogenase / metabolism*
  • Succinic Acid / chemistry
  • Succinic Acid / metabolism

Substances

  • Fumarates
  • Succinic Acid
  • Malate Dehydrogenase
  • Succinate Dehydrogenase
  • Fumarate Hydratase