A transition-metal chromophore as a new, sensitive spectroscopic tag for proteins: selective covalent labeling of histidine residues in cytochromes c with chloro(2,2':6',2″-terpyridine)platinum(II) chloride
A transition-metal chromophore as a new, sensitive spectroscopic tag for proteins: selective covalent labeling of histidine residues in cytochromes c with chloro(2,2':6',2″-terpyridine)platinum(II) chloride
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过渡金属发色团作为蛋白质的新型、灵敏光谱标签：用氯(2,2:6,2"-三联吡啶)氯化铂(II)选择性共价标记细胞色素c中的组氨酸残基
DOI：
10.1021/ja00249a023
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发表时间：
1987
影响因子：
15
通讯作者：
N. Kostić
中科院分区：
文献类型：
--
作者：
E. Ratilla;N. Kostić
以马和金枪鱼细胞色素c为例，研究了铂(Trpy)Cl/sup+/对蛋白质的反应活性和选择性。新的过渡金属试剂在pH为5时对组氨酸残基具有专一性。咪唑一步取代氯/超-/配体，产率较高。通过UV-Vis分光光度法和肽图谱实验鉴定了马蛋白中的His 26和His 33以及金枪鱼蛋白中的His 26结合部位。制备并表征了咪唑、组氨酸、组氨酸衍生物和含有组氨酸的多肽的模型配合物。共价连接的铂(Trpy)/sup~(2+)/标记物可以很容易地通过阳离子交换层析分离蛋白质衍生物。UV-Vis光谱、循环伏安法、差示脉冲伏安法、EPR波谱和/sup 1/H核磁共振波谱表明，这些标记不干扰细胞色素c的构象和还原电位。铂(Trpy)Cl/sup+/的选择性与PtCl/sub4//sup2-/完全相反，尽管它们都是铂(II)-氯络合物。由于三联吡啶配体的空间效应和电子效应之间的相互作用，新试剂对蛋氨酸(硫代醚)和胱氨酸(二硫键)不起反应，否则这两种配体是高度亲核的配体，但对咪唑非常活性，否则是相对更弱的配体。预先形成的过渡金属络合物对蛋白质的不寻常和有用的选择性显然可以通过明智地选择辅助配体来实现。
Reactivity and selectivity of Pt(trpy)Cl/sup +/ toward proteins are studied with cytochromes c from horse and tuna as examples. The new transition-metal reagent is specific for histidine residues at pH 5. The reaction, facile one-step displacement of the Cl/sup -/ ligand by imidazole, produces good yield. The binding sites, His 26 and His 33 in the horse protein and His 26 in the tuna protein, are identified by UV-vis spectrophotometry and by peptide-mapping experiments. Model complexes with imidazole, histidine, histidine derivatives, and histidine-containing peptides are prepared and characterized. The covalently attached Pt(trpy)/sup 2 +/ labels allow easy separation of the protein derivatives by cation-exchange chromatography. The labels do not perturb the conformation and reduction potential of cytochrome c, as shown by UV-vis spectrophotometry, cyclic voltammetry, differential-pulse voltammetry, EPR spectroscopy, and /sup 1/H NMR spectroscopy. The selectivity of Pt(trpy)Cl/sup +/ is entirely opposite from that of PtCl/sub 4//sup 2 -/ although both of them are platinum(II)-chloro complexes. Owing to an interplay between the steric and electronic effects of the terpyridyl ligand, the new reagent is unreactive toward methionine (a thio ether) and cystine (a disulfide), which are otherwise highly nucleophilic ligands, but very reactive toward imidazole, which is otherwise a relativelymore » weak ligand. Unusual and useful selectivity of preformed transition-metal complexes toward proteins evidently can be achieved by a judicious choice of ancillary ligands.« less