Resonance Raman Spectroscopy of Intact Mitochondria

完整线粒体的共振拉曼光谱

基本信息

批准号：
13640501
负责人：
OGURA Takashi
金额：
$ 1.92万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Mitochondria was isolated from porcine heart and the fraction that had a respiratory control ratio higher than 3 was used for Raman measurements when succinate was used as respiratory substrate. By addition of succinate, resonance Raman spectra of reduced mitochondria was measured, which exhibited vibrational spectra of hemes in cytochromes. By flushing CO we observed new Raman bands at 517, 576 and 369 cm^<-1>. They showed downshifts upon ^<13>C^<18>O substitution. Accordingly, the band at 517 was assigned to the V_<Fe-CO> mode and the bands at 576 and 369 cm^<-1> to the modes associated with δ_<Fe-C-O> of cytochrome a_3 in cytochrome c oxidase. These frequencies are the same with those of corresponding modes of solubilized enzyme. This fact implies that the heme pocket structure of cytochrome a_3 is not perturbed by solubilization.Time-resolved resonance Raman spectra of the reaction of reduced mitochondria with oxygen were measured at a delay time of 0.6 ms. Raman bands at 571 and 804 cm^<-1> were observed and they showed downshifts upon ^<18>O_2 substitution. Based on the result, we assigned the bands at 571 and 804 cm^<-1> to the V_<Fe-O2> mode of oxygenated and the V_<Fe=O> mode of oxo intermediate, respectively. The difference of the time evolution of these intermediates between solubilized enzyme and intact mitochondria will be examined in the next step.The reaction of solubilized enzyme in the mixed valence state with oxygen was studied with time-resolved resonance Raman spectroscopy. The decay of the oxygenated intermediate took place synchronously with the rise of the oxo intermediate and the decay of the oxygenated intermediate was significantly slower at alkaline pH than at neutral pH. These data for solubilized enzyme are important to analyze the reaction of intact mitochondria.

线粒体是从猪心中分离出来的，当将琥珀酸盐用作呼吸底物时，其呼吸控制比高于3的馏分被用于拉曼测量。通过添加琥珀酸酯，测量了线粒体减少的共振拉曼光谱，从而暴露了细胞色素中血液的振动光谱。通过Flushing Co，我们在517、576和369 cm^<-1>上观察到了新的拉曼乐队。他们在 ^<13> c ^<18> o取代时显示了降档。根据，将517处的频带分配到V_ <fe-CO>模式，并在576和369 cm^<-1>的369 cm^<-1>中分配给与细胞色素c氧化酶中细胞色素A_3相关的模式。这些频率与那些相应的可溶酶模式相同。这一事实意味着，细胞色素A_3的血红素袋结构不会受到溶液的干扰。在0.6 ms的延迟时间为0.6 ms，测量了线粒体与氧的反应的时间分解的共振拉曼光谱。观察到在571和804 cm ^<-1>处的拉曼带，并在替代时显示降档。基于结果，我们分别将571和804 cm^<-1>的频带分配给了氧气的V_ <fe-O2>分别分别为Oxo中间体的V_ <fe = O>模式。这些中间体在可溶性酶和完整线粒体之间的时间演变的差异将在下一步中进行检查。与氧气中的混合价状态下可溶性酶与氧气的反应与时间分解的分辨率拉曼光谱研究了。含氧中间体的衰减与Oxo中间体的上升同步发生，而在酗酒线pH时，含氧中间体的衰减明显慢于中性pH值。这些可溶性酶的数据对于分析完整线粒体的反应很重要。