High-pressure liquid chromatography quantitation of cytochrome c using 393 nm detection |
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Authors: | Picklo M J Zhang J Nguyen V Q Graham D G Montine T J |
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Affiliation: | Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. matthew.picklo@mcmail.vanderbilt.edu |
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Abstract: | The release of cytochrome c from the mitochondrial intermembrane space can induce apoptotic cell death. Previous methods to detect cytochrome c release from mitochondria have relied upon immunoblotting, a procedure that can be limited by nonlinearity of signal, epitope masking, and impracticality for large numbers of samples. In order to circumvent these limitations, we have developed a reverse-phase high-pressure liquid chromatography method for cytochrome c detection and quantitation by taking advantage of a novel acid-induced absorbance maximum at 393 nm for cytochrome c in buffer containing 0.1% trifluoroacetic acid. Using a C4 reverse-phase analytical column, this assay had a quantitation limit of 10 ng (0.8 pmol) of cytochrome c. We demonstrated the detection and quantitation of cytochrome c from isolated mitochondria. This method of cytochrome c analysis may be useful for the study of agents that cause mitochondrial dysfunction and apoptotic cell death. |
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