Assay of succinate dehydrogenase activity by the tetrazolium method: Evaluation of an improved technique in skeletal muscle fractions

Abstract

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspensions incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5