Capillary zone electrophoretic determination of cytochrome c in mitochondrial extracts and cytosolic fractions:: Application to a digitalis intoxication study

We describe a capillary zone electrophoretic procedure with photodiode-array detection for the determination of the apoptogenic protein cytochrome c in cytosolic fractions and mitochondrial extracts from guinea pig hearts. Optimal separation was achieved with a 100 mM phosphates electrolyte solution at pH 2.05. The applied voltage was 25 kV and the capillary temperature was kept constant at 25 +/- 0.5 degrees C. The method was linear over the concentration range of 0.2-600 pM. All determination coefficients were higher or equal to 0.9989. Limits of detection and quantitation were 0.06 pM (S/N = 3) and 0.21 pM (S/N = 10), respectively. The present method offers a time-saving way to determine cytochrome c since it can be completed in 12 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedure is illustrated by experiments that quantify cytochrome c released under control conditions and in a digitalis intoxication experimental animal model, in which cytochrome c content was successfully determined and was found to be (mean +/- standard deviation): control cytosol (0.48 +/- 0.01 pM), digitalis-intoxicated cytosol (0.85 +/- 0.01 pM), control mitochondria (1.11 +/- 0.1 pM) and digital is-intoxicated mitochondria (0.75 +/- 0.02 pM). Recovery results ranged from 98.4 to 110.2%. Hence, the proposed analytical method could be useful to elucidate the digitalis intoxication mechanism as well as the role of cytochrome c in mediating apoptosis. (C) 2007 Elsevier B.V. All rights reserved.