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Molecular motion of metal-free and metal-substituted cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal-free cytochrome c is around 10 ns. We find that the anisotropy of fluorescence of metal-free cytochrome c when bound to mitochondria does not decay, but when bound to phospholipids has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of zinc cytochrome c, as measured by triplet-triplet absorption and phosphorescence emission is z 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane-bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 ps and the slow component with relaxation time of z 6 ms.The slow component appears to be due to the rotation of the entire mitochondrion, whereas the fast component was interpreted to be due to the rotation of cytochrome c in a cone about a single axis perpendicular to the plane of the membrane surface.The question of organization of the mitochondrial electron transfer components is fundamental to the understanding of electron transfer and ATP coupling. There are several lines of evidence that the high rates and specificity of electron transfer in mitochondria is achieved through precise structural organization in terms of location and orientation of the components of the mitochondrial respiratory chain. For example, the hemes and iron-sulfur centers appear to be oriented with respect to the plane of the membrane [I -31.On the other hand, the possibility of lateral diffusion of the membrane proteins is an important aspect in understanding the electron transport mechanism. The electron transfer in the respiratory chain of mitochondria between the reductase and the oxidase, and the role of cytochrome c in the process presents an interesting question concerning the motion and the organization of the integral proteins. Cytochrome c is a small peripheral membrane protein which provides a path for electron transfer between two large intrinsic membrane proteins, the cytochrome bcl complex and the cytochrome oxidase. Because of this relationship, an intriguing question is raised: what type and what degree of molecular motion are required for the interaction of cytochrome c with both the oxidase and the reductase? There are several reports in the literature indirectly implying the motion of integral proteins [4-61. In this paper we discuss several types of molecular motion (whole molecule and molecular structural fluctuation) in metal-free and metal-substituted cytochrome c, utilizing the anisotropy of emissi...

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We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles. Optimum conditions with respect to detergent concentration, time, sample size, and yield are discussed. The method's utility in downstream analyses and ease of processing multiple samples simultaneously is also described. All the reagents in this method can be assembled in-house, are economical, and are comparable, if not superior, to commercially available kits in terms of mitochondrial yield and integrity. • Rapid enrichment of mitochondria from mammalian cells using commonly available reagents. • Multiple samples can be processed simultaneously. • Works over a wide range of sample size (1 million to 100 million cells).