A new measurement system for UV resonance Raman spectra of large proteins and its application to cytochrome c oxidase
A new measurement system for UV resonance Raman spectra of large proteins and its application to cytochrome c oxidase
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DOI：
10.1021/jp000357p
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发表时间：
2000-11-23
影响因子：
3.3
通讯作者：
Kitagawa, T
中科院分区：
文献类型：
--
作者：
Aki, M;Ogura, T;Kitagawa, T
提出了一种适用于少量大蛋白质样品的新型紫外共振拉曼（UVRR）测量系统，并给出了该系统在牛细胞色素c氧化酶（CcO）中的应用结果。为了最大限度地减少高通量紫外激光照射造成的样品损伤，并拒绝从样品中可见荧光，锁模Ar+离子激光器和日盲多通道检测器的倍频，分别采用。设计了一个新的旋转池，以便在旋转池期间可以搅拌样品溶液。所有这些设备的组合导致成功地观察到高质量的UVRR光谱的CcO在244 nm激发。色氨酸和酪氨酸残基的RR带占主导地位的观察到的光谱，而一个额外的带出现在1656 cm（-1）。额外带的频率以及所有其他带的频率不受金属中心和配体与血红素a结合的氧化还原变化的影响（3）。在pH 9.1的静息状态下检测到具有低pK（a）值的酪氨酸残基的去质子化。检查所有可能的分配，使我们得出结论，额外的条带产生的亚油酰基侧链的磷脂和其强度表明存在21个亚油酰基基团每个CcO分子。
A new type of ultraviolet resonance Raman (UVRR) measurement system suitable to a limited amount of large protein samples is proposed and the results from its application to bovine cytochrome c oxidase (CcO) is presented. To minimize the sample damage caused by high-flux UV laser illumination and to reject visible fluorescence from the sample, frequency-doubling of a mode-locked Ar+ ion laser and a solar blind multichannel detector were employed, respectively. A new spinning cell was designed so that the sample solution could be stirred during spinning of the cell. Combination of all these devices resulted in successful observation of high quality UVRR spectra of CcO excited at 244 nm. The RR bands of tryptophan and tyrosine residues dominated the observed spectra, while an extra band appeared at 1656 cm(-1). The frequency of the extra band as well as those of all other bands were unaltered by the redox change of metal centers and ligand binding to heme a(3). Deprotonation of a tyrosine residue(s) with a low pK(a) value was detected for the resting state at pH 9.1. Examination of all possible assignments led us to conclude that the extra band arose from the linoleoyl side chain of phospholipids and its intensity suggested the presence of 21 linoleoyl groups per CcO molecule.