Succinate dehydrogenase (SDH) activity assay: principle, protocol and calculations

Succinate dehydrogenase sits in the inner mitochondrial membrane and is the only enzyme that belongs to both the Krebs (TCA) cycle and the electron transport chain. It oxidises succinate to fumarate, reducing its FAD cofactor, and passes the electrons through iron-sulfur clusters to ubiquinone. In the electron transport chain it is called complex II.

Because it links the two pathways, SDH activity is a direct measure of mitochondrial function. It is measured in metabolism and mitochondrial disease research, toxicology, exercise and muscle physiology, tumour biology, where SDH subunit mutations act as tumour suppressor defects, and plant and microbial physiology.

SDH’s natural electron acceptor, ubiquinone, cannot be seen. Colorimetric assays supply an artificial acceptor that changes colour when reduced:

Kits usually add inhibitors of complexes III and IV, such as antimycin A or azide, and of complex I, such as rotenone, so electrons go to the dye instead of onward through the chain.

• Isolated mitochondria give the cleanest signal. Homogenise tissue in cold isolation buffer, remove nuclei and debris at about 600–1,000 × g, then pellet mitochondria at about 8,000–12,000 × g.
• Tissue homogenates and cell lysates work with most kits and are simpler. Homogenise on ice in the kit’s extraction buffer, centrifuge and keep the supernatant or resuspended pellet as directed.
• Access to the enzyme. Succinate has to reach SDH, so some protocols permeabilise membranes with a freeze-thaw cycle or a mild detergent. Follow the kit, and keep the method the same across samples.
• Freshness. Assay the same day when you can. If not, snap-freeze and store at -80 °C, and test whether storage changes activity.

Measure total protein in the same extract with a compatible assay so activity can be expressed per mg of protein. The lysis buffer guide explains which buffers keep enzymes active.

The outline below shows the logic. Use the volumes and times in your kit manual.

1. Bring the assay buffer to the assay temperature, often 25 or 37 °C.
2. In a clear 96-well plate or cuvette, combine buffer, inhibitors, DCPIP and the electron carrier.
3. Add the sample. Set up a parallel well without succinate as the sample blank.
4. Start the reaction by adding succinate, mixing well.
5. Read absorbance at 600 nm every 20–30 seconds for 3–10 minutes.
6. Use the linear part of the curve to calculate the rate, ΔA per minute.

Subtract the blank rate from the sample rate, then convert the absorbance change into moles of dye reduced using the Beer-Lambert law:

Activity (µmol/min/mL) = (ΔA/min × V_total) ÷ (ε × l × V_sample)

• ΔA/min: blank-corrected change in absorbance per minute
• V_total and V_sample: reaction and sample volumes in the same units
• ε: the extinction coefficient in mM⁻¹ cm⁻¹ given in your kit manual. For DCPIP at 600 nm it is about 19–21, depending on pH.
• l: path length in cm. It is 1 cm in a cuvette but less in a microplate well, so correct for it or use the kit’s standard

Divide by protein concentration to report µmol/min per mg of protein. One unit (U) is usually defined as 1 µmol of substrate converted, or dye reduced, per minute.

Soil dehydrogenase assays use a related idea: microbial dehydrogenases reduce a tetrazolium salt such as TTC to a red formazan, which makes total dehydrogenase activity a standard indicator of soil microbial activity. For other enzymes, the enzyme activity assay guide lists reactions and readouts, and the biochemical assay range includes SDH and related kits.