Picklo M J, Zhang J, Nguyen V Q, Graham D G, Montine T J
Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Anal Biochem. 1999 Dec 15;276(2):166-70. doi: 10.1006/abio.1999.4349.
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.
细胞色素c从线粒体膜间隙的释放可诱导细胞凋亡性死亡。以往检测细胞色素c从线粒体释放的方法依赖于免疫印迹法，该方法可能受到信号非线性、表位掩盖以及不适用于大量样本等限制。为了规避这些限制，我们利用细胞色素c在含0.1%三氟乙酸的缓冲液中在393 nm处出现的新型酸诱导吸光度最大值，开发了一种反相高压液相色谱法用于细胞色素c的检测和定量。使用C4反相分析柱，该检测方法对细胞色素c的定量限为10 ng（0.8 pmol）。我们展示了从分离的线粒体中检测和定量细胞色素c的方法。这种细胞色素c分析方法可能有助于研究导致线粒体功能障碍和细胞凋亡性死亡的因素。
