Malate Dehydrogenase Assay

Malate dehydrogenase (MDH2) catalyzes the last reaction of the TCA or Krebs cycle, converting malate and NAD+ to oxaloacetate and NADH in the mitochondria. In reversing the reaction, MDH is also involved in gluconeogenesis, allowing oxaloacetate/malate to leave the mitochondria. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic MDH or MDH1, which is followed by conversion of oxaloacetate to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK). The interconversion between malate and oxaloacetate also constitutes the essential steps of the malate-aspartate shuttle. This colorimetric assay is based on malate dehydrogenase-catalyzed oxidation of malate, where the resulting NADH can then convert a nearly colorless probe to a colored product; the intensity of the colored product is proportional to the amount of MDH in the sample, exhibiting maximum absorbance at 440nm.