Synthesis and spectral characterization of sulfhydryl-reactive fluorescent probes.
Synthesis and spectral characterization of sulfhydryl-reactive fluorescent probes.
复制标题
巯基反应性荧光探针的合成和光谱表征。
DOI：
10.1006/abio.1996.0087
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Cremo,CR
中科院分区：
文献类型：
--
作者：
Pouchnik,DJ;Laverman,LE;Janiak-Spens,F;Jameson,DM;Reinhart,GD;Cremo,CR
We synthesized two sulfhydryl-reactive fluorescent probes, Br-ANT (2-amino-benzoic acid, 2-(bromoace- tyl)hydrazide) and Br-MANT (N-[2-[(bromoacetyl)amino]ethyl]-2-(methylamino)benzamide). Br-ANT and Br-MANT contain an anthraniloyl andN-methyl anthraniloyl group, respectively, linked to the sulfhydryl-reactive bromoacetyl moiety. The cysteine adducts have absorption maxima at 323 and 326 nm, with molar extinction coefficients of 2100 and 2900M−1cm−1, for Br-ANT and Br-MANT, respectively, making these probes excellent acceptors for tryptophan. The absorption spectra and quantum yields were constant at pH levels useful for protein studies (pH 5–8). Quantum yields of Br-ANT and Br-MANT were 0.16 and 0.42, emission maxima were 432 and 440 nm, and fluorescence lifetimes were 1.3 and 7.8 ns, respectively. The emission of Br-ANT-Cys and Br-MANT-Cys shifted to shorter wavelengths with decreasing solvent polarity. Polarization values were maximal between 330 and 375 nm. Both probes reacted selectively and stoichiometrically with the single cysteine residue of a model protein. The labeled protein exhibited relatively long lifetimes (9–10 ns), suggesting that these probes will be generally useful for rotational studies.
