A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation

Ferricyanide reduction frequently is analyzed to determine the activity of membraneous reductases. An improved, highly sensitive, and rapid method for quantitative endpoint determination of ferrocyanide is presented. Ferrocyanide is oxidized by Fe3+ in the presence of Ferene-S under acid conditions to form a chromogenic Ferene-S/Fe2+ complex. The latter is quantitated at 593 nm with a sensitivity of 33.2 mM−1 · cm−1. The assay is 60% more sensitive to ferrocyanide (and with a 50% lower detection limit) than the prevailing method of Avron and Shavit, which employs sulfonated bathophenanthroline as the ferrous chromogen. Both pH dependence and potential sources of interference are discussed. Using the method, a sulfhydryl-sensitive, ascorbate-stimulated transplasma membrane ferricyanide reductase was assayed in human chronic myeloid (K562) leukemia cells. Furthermore, malonate-sensitive succinate dehydrogenase activity of heart mitochondria was easily assayed with ferricyanide as terminal electron acceptor. The current method will suit routine applications demanding high throughput, robustness, and sensitivity in a 96-well plate format.