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 100mM phosphates electrolyte solution at pH 2.05. The applied voltage was 25kV and the capillary temperature was kept constant at 25+/-0.5 degrees C. The method was linear over the concentration range of 0.2-600pM. All determination coefficients were higher or equal to 0.9989. Limits of detection and quantitation were 0.06pM (S/N=3) and 0.21pM (S/N=10), respectively. The present method offers a time-saving way to determine cytochrome c since it can be completed in 12min, 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+/-0.01pM), digitalis-intoxicated cytosol (0.85+/-0.01pM), control mitochondria (1.11+/-0.1pM) and digitalis-intoxicated mitochondria (0.75+/-0.02pM). 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.
del Valle-Mondragón
Leonardo
L
Departamento de Farmacología, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1, Sección XVI, 14080 Tlalpan, Mexico DF, Mexico.
Ramírez-Ortega
Margarita
M
Zarco-Olvera
Gabriela
G
Sánchez-Mendoza
Alicia
A
Pastelín-Hernández
Gustavo
G
Tenorio-López
Fermín Alejandro
FA
D000818:Animals; D015153:Blotting, Western; D045304:Cytochromes c / Q000032:analysis*; D003600:Cytosol / Q000201:enzymology*; D004070:Digitalis / Q000506:poisoning*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology*; D012680:Sensitivity and Specificity
D045304:Cytochromes c