Gomez B, Robinson N C
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, 78284-7760, USA.
Anal Biochem. 1999 Feb 1;267(1):212-6. doi: 10.1006/abio.1998.2998.
Quantitative determination of cardiolipin from two mitochondrial electron-transferring complexes was achieved using a rapid and sensitive silicic acid HPLC method combined with digital analysis of the elution profile. Phospholipid samples containing as little as 0. 01 nmol of cardiolipin were accurately analyzed. Phospholipids from detergent-solubilized cytochrome bc1 (EC 1.10.2.2) and cytochrome c oxidase (EC 1.9.3.1) were extracted by an organic two-phase system and analyzed by isocratic normal-phase HPLC after dissolving the dried sample in the mobile phase (cyclohexane:2-propanol:5 mM phosphoric acid, 50:50:2.9, v/v/v). Analysis was performed by the method of standard addition in which increasing amounts of cardiolipin (0 to 5 nmol) are added to a constant amount of phospholipid extract containing an unknown amount of cardiolipin. By determining the slope and intercept of a plot of the HPLC elution peak area as a function of the amount of standard cardiolipin added, the amount of cardiolipin in the unknown is determined. By this analysis, purified, detergent-solubilized bovine heart cytochrome bc1 and cytochrome c oxidase contained 9.2 +/- 0.7 and 3.05 +/- 0.05 mol cardiolipin per mole of enzyme, respectively. The method was also used to prove that cardiolipin could be completely removed from each complex by digestion with Crotalus atrox phospholipase A2, i.e., each delipidated complex contained less than 0.05 mol cardiolipin per mole of complex. The rapidity and high sensitivity of this method make it very useful for analysis of cardiolipin in other biological samples.
采用快速灵敏的硅酸高效液相色谱法并结合洗脱图谱的数字分析，实现了对两种线粒体电子传递复合物中心磷脂的定量测定。含有低至0.01 nmol心磷脂的磷脂样品得到了准确分析。通过有机两相系统提取去污剂溶解的细胞色素bc1（EC 1.10.2.2）和细胞色素c氧化酶（EC 1.9.3.1）中的磷脂，将干燥样品溶解于流动相（环己烷：2-丙醇：5 mM磷酸，50:50:2.9，v/v/v）后，通过等度正相高效液相色谱进行分析。采用标准加入法进行分析，即向含有未知量心磷脂的恒定磷脂提取物中加入递增剂量的心磷脂（0至5 nmol）。通过确定高效液相色谱洗脱峰面积随标准心磷脂添加量变化的曲线的斜率和截距，确定未知样品中心磷脂的含量。通过该分析，纯化的、去污剂溶解的牛心脏细胞色素bc1和细胞色素c氧化酶每摩尔酶分别含有9.2±0.7和3.05±0.05摩尔心磷脂。该方法还用于证明通过用矛头蝮蛇磷脂酶A2消化可将心磷脂从每种复合物中完全去除，即每种脱脂复合物每摩尔复合物含有少于0.05摩尔心磷脂。该方法的快速性和高灵敏度使其在分析其他生物样品中的心磷脂时非常有用。
