Colorimetric determination of succinic acid using yeast succinate dehydrogenase

An enzymatic method for the rapid determination of succinic acid in biological fluids was developed utilizing yeast mitochondria as a source of succinate dehydrogenase. The yeast enzyme catalyzes a complete stoichiometric reduction of 2- (p-iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium chloride to a red formazan. The formazan is extracted into ethylacetate and its absorbance measured at 490 nm. The method is simple, specific, reproducible, and very sensitive (0.01 to 0.14 mumol). The yeast enzyme can be stored in liquid nitrogen for periods of at least 30 days with no significant change in specific activity. In this respect it is superior to a variety of succinate dehydrogenase preparations from animal tissues. The method was applied to measurement of succinic acid excreted by nonproliferating yeast cells metabolizing glucose. Derepressed yeast cells secreted several-fold as much succinic acid as repressed cells submitted to identical test conditions.