Categorized: Biochemical Assays
All dehydrogenase assays are preformed in the same way, although the electron donor (eg., succinate), the electron acceptor(s) and the final chromagenic substrate (eg., DCIP) must be varied according to the energy state of the dehydrogenase. Dehydrogenase enzyme assays are used by us as markers of the inner membrane.
- Add the following to sample to be tested and incubate at 25oC for six minutes. You will probably need to test a range of sample concentrations, but just do one at a time.
2 ml 0.1M Tris-HCl, pH 8.0 0.1 ml 0.2M KCN (13 mg/ml), freshly prepared 0.1 ml 0.6M sodium succinate (162 mg/ml) 10 µl enzyme or membrane preparation 0.64 ml distilled water
- Add 0.1 ml of freshly prepared 12.5mM phenazine methosulphonate (3.83mg/ml) and 0.05ml freshly prepared 2.5mM dichloroindole phenol (0.73mg/ml, DCIP) and mix briefly.
- Measure the change in OD600 using the spectrophotometer hooked up to the chart recorder. The blank contains no enzyme or membrane preparation.
- Calculate the amount of enzyme using the appropriate molar extinction coefficient of the final elector acceptor (DCIP) reduction. For DCIP, this equals 22,000 l/mole.
- Express the enzyme activity as umoles DCIP reduced/minutes/milligram of protein.