A simplified and reliable assay for complex I in human blood lymphocytes
Complex I activity of the mitochondrial respiratory chain is difficult to measure in blood lymphocytes because of the limited access of substrates to the enzyme complex in these cells. The results of the present study show that permeabilization of human blood lymphocytes in the presence of protease inhibitors by three cycles of freeze-thawing enables reproducible detection of the rotenone-sensitive complex I activity. To that end, the water-soluble coenzyme Q10analogue CoQ1and a relatively high concentration of blood lymphocytes were combined in small quartz cuvettes so that the amount of blood needed for this assay remained low. The relationship between the initial rate of NADH oxidation by complex I and the protein concentration was quasi-linear. The fractional inhibition of the total NADH:CoQ1oxidoreductase by a saturating concentration of rotenone decreased sharply at CoQ1concentrations higher than 20 μM, which is indicative, but does not prove the involvement of a second CoQ1binding site at complex I. Since the present complex I assay requires only a small amount of blood, the functionality of this important respiratory chain complex can be assessed in an easy and reliable manner not only in adult patients but also in children suspected to have a mitochondrial disease.
|Keywords||Mitochondria, Oxidative phosphorylation (OXPHOS), Respiratory chain, Rotenone-insensitive NADH:coenzyme Q oxidoreductase (RINQ), Rotenone-sensitive NADH:coenzyme Q oxidoreductase (complex I)|
|Persistent URL||dx.doi.org/10.1016/j.jim.2007.07.009, hdl.handle.net/1765/35195|
de Wit, L.E.A., Spruijt, L., Schoonderwoerd, G.C., de Coo, I.F.M., Smeets, H.J., Scholte, H.R., & Sluiter, W.. (2007). A simplified and reliable assay for complex I in human blood lymphocytes. Journal of Immunological Methods, 326(1-2), 76–82. doi:10.1016/j.jim.2007.07.009