The complex of mitochondrial protein cytochrome c (CytC) with anionic phospholipid cardiolipin (CL) plays a crucial role in the initiation of apoptosis by catalyzing lipid peroxidation in mitochondrial membranes. In our previous papers, we found that CytC and CL mixed in millimolar concentrations form a sediment showing microcrystals composed of nanospheres (Cyt-CL) of 11–12 and 8 nm in diameter. The hypothesis was proposed that Cyt-CL, having hydrophobic shell, may appear inside the membrane lipid bilayer in mitochondria and peroxidase membrane phospholipids so initiating the apoptotic cascade. In this work, Cyt-CL complex dissolved in chloroform or hexane was investigated as a model of the complex in mitochondrial membranes. We used dynamic light scattering method to measure the size of the particles. The analysis of particles size distribution of Cyt-CL in chloroform allows to reveal three dominant diameters of 12.1 ± 1.4, 7.8 ± 1.0, and 4.7 ± 0.7 nm. The first two values are closed to those, earlier obtained with small-angle X-ray scattering method in Cyt-CL microcrystals, 11.1 ± 1.0 and 8.0 ± 0.7 nm. CL extracted in chloroform-methanol forms a real solution of particles with diameter of 0.7 ± 0.1 nm. In methanol-water phase, CL and CL + CytC mixture form particles of 83.7 ± 9.8 and 71.3 ± 11.6 nm, respectively. Apparently, cardiolipin in 50% methanol forms single-layer liposomes regardless of the presence of CytC in the medium. Partial unfolding of CytC in the complex was evidenced by (a) appearance of fluorescence of tyrosine and tryptophan residues and (b) disappearance of the absorption band at 699 nm due to breakdown of heme iron – methionine bond > F⋯S(Met80). In hydrophobic solvent Cyt-CL exhibited quasi-lipoperoxidase and lipoxygenase activity as was shown in kinetic measurements of chemiluminescence enhanced by coumarin C-525, a selective sensitizer of chemiluminescence, associated with reactions of lipid peroxyl radicals.

Our data in this model system do not contradict the hypothesis (Vladimirov, Y.A. et al. Biochemistry (Mosc) 78, 1086–1097) that nanospheres of Cyt-CL complex, embedded into the lipid phase of mitochondrial membrane, catalyze lipid peroxidation, thereby initiating apoptosis.

线粒体蛋白细胞色素c的复合物（CytC）和阴离子磷脂心磷脂（CL）通过催化线粒体膜中的脂质过氧化作用，在凋亡的启动中起关键作用。在我们之前的论文中，我们发现以毫摩尔浓度混合的CytC和CL形成沉积物，显示出由直径为11–12且直径为8 nm的纳米球（Cyt-CL）组成的微晶。提出的假设是，具有疏水性壳的Cyt-CL可能会出现在线粒体的膜脂质双层内部和过氧化物酶膜磷脂中，从而引发凋亡级联反应。在这项工作中，研究了溶解在氯仿或己烷中的Cyt-CL复合物作为线粒体膜中复合物的模型。我们使用动态光散射法来测量颗粒的尺寸。分析Cyt-CL在氯仿中的粒径分布，可以揭示三个主要直径，分别为12.1±1.4、7.8±1.0和4.7±0.7 nm。前两个值与先前在Cyt-CL微晶中通过小角度X射线散射法获得的值接近11.1±1.0和8.0±0.7 nm。用氯仿-甲醇萃取的CL形成直径为0.7±0.1 nm的真实颗粒溶液。在甲醇-水相中，CL和CL + CytC混合物分别形成83.7±9.8和71.3±11.6 nm的颗粒。显然，不管培养基中是否存在CytC，在50％甲醇中的心磷脂都会形成单层脂质体。（a）酪氨酸和色氨酸残基的荧光出现，以及（b）由于血红素铁-蛋氨酸键> F⋯S（Met80）的分解，在699 nm处的吸收带消失，证明了复合物中CytC的部分展开。在疏水溶剂中，Cyt-CL表现出准脂过氧化物酶和脂氧合酶活性，如通过香豆素C-525（一种化学发光的选择性敏化剂）增强的化学发光的动力学测量所显示的，该动力学与脂质过氧化自由基的反应有关。

我们在该模型系统中的数据与假设（Vladimirov，YA et al。Biochemistry（Mosc）78，1086-1097）并不矛盾，即Cyt-CL复合物的纳米球嵌入线粒体膜的脂质相中，催化脂质过氧化，从而启动细胞凋亡。