A colorimetric method for the estimation of succinic dehydrogenase activity.

Many methods for the measurement of succinic dehydrogenase activity have been proposed (l-20). These have varied from measurement of the entire system of electron transport with oxygen as the final acceptor (5, 21), to the determination of succinic dehydrogenase activity itself (6, 9, 22). The assay of succinic dehydrogenase alone was made possible by the finding that phenazine methosulfate can act as an intermediate electron carrier between this dehydrogenase and molecular oxygen. That phenazine methosulfate can act similarly as an electron carrier to tetrazolium salts, such as 2,2’, 5,5’-tetraphenyl-3,3’-(3,3’dimethoxy-4,4’-biphenylene) ditetrazolium chloride, was shown in the histochemical preparations of Farber and Bueding (23). The stability of the formazans formed on reduction of tetrazolium salts has been used advantageously in the development of calorimetric methods for succinic dehydrogenase (16-20). However, the insoluble nature of the formazans produced upon reduction has made necessary the extraction of the pigment into an organic solvent for calorimetry. The observation that some formazans exhibit substantive properties (24) suggested that the addition of a simple protein into the reaction mixture might keep the formazan dispersed finely enough to permit determination of the color density in aqueous solution. Gelatin served very well for this purpose, since it did not alter enzymatic activity. Although some of the tetrazoles that have been employed either were not readily reduced by succinoxidase or could not compete sufficiently well with oxygen for the enzymatically liberated electrons, so that anaerobiosis was required (16-l@, previous work led to the recognition that the sensitivity of the tetrazole or the ease of its reduction could be markedly influenced by certain electronegative groups attached to either the N-2 phenyl or N-3 phenyl rings (25-27). Thus, INT’ was found to be reduced much more rapidly than its unsubstituted triphenyl analogue (20,25,27,28). Similarly, nitro-BT was a much more sensitive histochemical indicator of succinic dehydrogenase activity than its analogue, BT, which lacked the nitro moiety (24, 29). In addition to their more rapid reduction, nitro-BT and INT competed satisfactorily with molecular oxygen, thus elimi-