Lemke H D, Oesterhelt D
Eur J Biochem. 1981 Apr;115(3):595-604. doi: 10.1111/j.1432-1033.1981.tb06244.x.
Treatment of the purple membrane with tetranitromethane under controlled conditions leads to the nitration of 3 mol tyrosine/mol bacteriorhodopsin. The combination of subtilisin digestion and cyanogen bromide cleavage with subsequent analysis of the resulting peptide mixture by high-performance liquid chromatography, allows identification of the positions modified in the polypeptide chain. Tyrosines 26 and 64 are fully nitrated, whereas tyrosines 131 and 133 are nitrated to about 60% and 40%, respectively. Reduction of the nitrated membranes with the water-soluble ionic agent dithionite leaves only tyrosine 26 nitrated indicating that the residues 64, 131 and 133 are located on the membrane surface. As a result of nitration, the purple complex shifts its absorption maximum from 568 nm to 532 nm. Dithionite reduction of the nitrated membrane does not reverse this effect. Removal of the retinal and reconstitution maintains the blue-shifted absorption of the chromophore. A pH-dependent equilibrium of the chromophore with a further red-shifted form is observed. The pK of this transition is at about pH 9. Because nitration of tyrosine leads to a drastic decrease of its pK a participation of tyrosine 26 in the chromophoric structure via a hydrogen bridge is suggested. This finding is consistent with a model of chromophore structure published earlier [U. Fischer and D. Oesterhelt (1979) Biophys. J. 31, 139--146].
在可控条件下用四硝基甲烷处理紫膜，会使每摩尔细菌视紫红质中有3摩尔酪氨酸发生硝化反应。枯草杆菌蛋白酶消化和溴化氰裂解相结合，随后通过高效液相色谱分析所得肽混合物，可确定多肽链中被修饰的位置。酪氨酸26和64被完全硝化，而酪氨酸131和133的硝化程度分别约为60%和40%。用可溶于水的离子试剂连二亚硫酸盐还原硝化后的膜，结果只有酪氨酸26仍处于硝化状态，这表明残基64、131和133位于膜表面。硝化作用导致紫色复合物的最大吸收波长从568 nm移至532 nm。用连二亚硫酸盐还原硝化后的膜并不能逆转这种效应。去除视黄醛并重新构建后，发色团仍保持蓝移吸收。观察到发色团与另一种红移形式之间存在pH依赖性平衡。这种转变的pK约为pH 9。由于酪氨酸的硝化会导致其pK大幅降低，因此推测酪氨酸26通过氢键参与发色团结构。这一发现与早期发表的发色团结构模型[U. Fischer和D. Oesterhelt（1979年），《生物物理杂志》31卷，139 - 146页]一致。
