High-pressure liquid chromatography quantitation of cytochrome c using 393 nm detection.
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.
Picklo
M J
MJ
Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. matthew.picklo@mcmail.vanderbilt.edu
Zhang
J
J
Nguyen
V Q
VQ
Graham
D G
DG
Montine
T J
TJ
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003574:Cytochrome c Group / Q000032:analysis*; D006736:Horses; D008928:Mitochondria / Q000737:chemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry
D003574:Cytochrome c Group