High-pressure liquid chromatography quantitation of cytochrome c using 393nm detection

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptotic cell death. Previous methods to detect cytochrome c releas e from mitochondria have relied upon immunoblotting, a procedure that can b e limited by nonlinearity of signal, epitope mashing, and impracticality fo r large numbers of samples. In order to circumvent these limitations, we ha ve developed a reverse-phase high-pressure liquid chromatography method for cytochrome c detection and quantitation by taking advantage of a novel aci d-induced absorbance maximum at 393 nm for cytochrome c in buffer containin g 0.1% trifluoroacetic acid. Using a C4 reverse-phase analytical column, th is assay had a quantitation limit of 10 ng (0.8 pmol) of cytochrome c. We d emonstrated the detection and quantitation of cytochrome c fi om isolated m itochondria. This method of cytochrome c analysis may be useful for the stu dy of agents that cause mitochondrial dysfunction and apoptotic cell death. (C) 1999 Academic Press.