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用7-氯-4-硝基苯并-2-恶唑-1,3-二唑对细菌视紫红质进行定点荧光修饰。

Site-directed fluorogenic modification of bacteriorhodopsin by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole.

作者信息

Allegrini P R, Sigrist H, Schaller J, Zahler P

出版信息

Eur J Biochem. 1983 May 16;132(3):603-8. doi: 10.1111/j.1432-1033.1983.tb07406.x.

Abstract

Site-directed covalent modification of bacteriorhodopsin is achieved by reacting the hydrophobic probe 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) at neutral pH with purple membranes. The bacteriorhodopsin fluorescence thus produced is specific for a nucleophilic group. The spectral properties of NBD-modified bacteriorhodopsin indicate covalent interaction of the probe with the nucleophilic epsilon-amino group of a lysine residue. Modification of tyrosine can be excluded. As demonstrated by polypeptide fragmentation and subsequent sequence analysis, NBD binding is confined to lysine 41 within the primary structure of bacteriorhodopsin. Collisional fluorescence quenching with iodide demonstrates that, in NBD-treated purple membranes, the covalently bound label is not accessible in the aqueous phase. A hydrophobic location for the introduced fluorophor is thereby implied.

摘要

通过使疏水探针7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂(NBD-Cl)在中性pH下与紫膜反应,实现了对细菌视紫红质的定点共价修饰。由此产生的细菌视紫红质荧光对亲核基团具有特异性。NBD修饰的细菌视紫红质的光谱特性表明探针与赖氨酸残基的亲核ε-氨基发生了共价相互作用。可以排除酪氨酸的修饰。如多肽片段化和后续序列分析所示,NBD结合仅限于细菌视紫红质一级结构中的赖氨酸41。用碘化物进行的碰撞荧光猝灭表明,在NBD处理的紫膜中,共价结合的标记物在水相中无法接近。由此暗示了引入的荧光团的疏水位置。

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