Dixit B P, Waring A J, Wells K O, Wong P S, Woodrow G V, Vanderkooi J M
Eur J Biochem. 1982 Aug;126(1):1-9. doi: 10.1111/j.1432-1033.1982.tb06737.x.
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 approximately equal to 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 mus and the slow component with relaxation time of approximately equal to 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.
利用单重态和三重态发射的各向异性研究了无金属和金属取代的细胞色素c衍生物的分子运动。由于无金属细胞色素c的荧光寿命约为10纳秒，荧光各向异性提供了一种在纳秒时间尺度上研究细胞色素c运动的方法。我们发现，无金属细胞色素c与线粒体结合时荧光各向异性不衰减，但与磷脂结合时存在一小部分独立于整个分子旋转而衰减的成分。磷光的使用将研究的时间尺度扩展到毫秒范围，因为通过三重态-三重态吸收和磷光发射测量，锌细胞色素c激发三重态的寿命在室温下对于游离锌细胞色素c约为9毫秒，对于线粒体膜结合的锌细胞色素c约为7毫秒。线粒体膜结合的锌细胞色素c磷光发射各向异性的衰减明显呈双相；快速成分对应于300微秒的旋转弛豫时间，慢速成分的弛豫时间约为6毫秒。慢速成分似乎是由于整个线粒体的旋转，而快速成分被解释为由于细胞色素c在垂直于膜表面平面的单轴周围的圆锥体中旋转。
