Succinate Dehydrogenase (SDH) DCPIP/PMS Assay

We assayed detergent solubilized SDH activity using a modification of the DCPIP/PMS assay with paired samples, substituting 0.015% lauryl maltoside for 0.1% Triton X-100 (23). As placing rotating magnetic stimulators alongside a cuvette, within a spectrophotometer, could cause sOMF field distortion and interference to electronic components we therefore utilized parallel incubations. Control and sOMF treated paired samples were prepared from a common reaction mixture, from which we removed aliquots, for spectroscopic assay.

Effect of rotenone: PBS supplemented with 0.015% lauryl maltoside, 1 mM sodium cyanide and 2 μg/ml rotenone was maintained at 37°C. A pre-assay incubation mixture of activated SDH was prepared by transferring 5 ml into a centrifuge tube and 10 mM succinate and 1 mg/ml of detergent solubilized RLM was added, at 37°C. After 10 minutes incubation phenazine methosulphate (PMS) and dichlorophenolindophenol (DCPIP) were added to a final concentration of 150 μM from freshly prepared DMSO-stock. The mixture was split into a pair of glass-walled, thermostatically controlled, 37°C, incubation chambers (Rank Bros O₂-electrode). Two 100 μl aliquots (duplicates) were removed from each chamber, at t=0. These aliquots were transferred onto 4-wells of a 96-well-plate and the 600 – 500 nm absorbance was immediately measured using a BioTek Synergy spectrophotometer. At t=5 min, the next paired duplicate samples were removed, and a four 5 min on and 5 min off sOMF cycle was applied to the assay mixture in one chamber. Duplicate samples were removed from each chamber at the end of each on/off cycle.