Seligman A M, Karnovsky M J, Wasserkrug H L, Hanker J S
J Cell Biol. 1968 Jul;38(1):1-14. doi: 10.1083/jcb.38.1.1.
A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion.
一种基于3,3'-二氨基联苯胺（DAB）氧化聚合成嗜锇反应产物来显示细胞色素氧化酶活性的新方法，在光学显微镜和电子显微镜下，相对于基于纳迪反应的方法，改进了该酶的定位。反应产物以非液滴形式出现，能更准确地描绘心脏、肝脏和肾脏线粒体中细胞色素氧化酶的定位。在电子显微镜制备中，发现过量的反应产物会溢出到嵴间空间以及线粒体内外限制膜之间的外腔中。这一发现表明细胞色素c的酶活性位于嵴间空间的内表面，即线粒体内膜的外表面。通过使用嗜锇双四唑盐TC-NBT，琥珀酸脱氢酶活性也定位于此部位。综合来看，呼吸链两部分的反应位点对米切尔的化学渗透假说具有重要意义，该假说提出了线粒体呼吸链电子传递过程中的能量守恒机制。
