Synthesis and spectral characterization of sulfhydryl-reactive fluorescent probes.
We synthesized two sulfhydryl-reactive fluorescent probes, Br-ANT (2-amino-benzoic acid, 2-(bromoacetyl)hydrazide) and Br-MANT (N-[2-[(bromoacetyl)amino]ethyl]-2-(methylamino)benzamide). Br-ANT and Br-MANT contain an anthraniloyl and N-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 2900 M-1 cm-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.
Pouchnik
D J
DJ
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Laverman
L E
LE
Janiak-Spens
F
F
Jameson
D M
DM
Reinhart
G D
GD
Cremo
C R
CR
D001549:Benzamides / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D062367:ortho-Aminobenzoates / Q000138:chemical synthesis* / Q000737:chemistry
C099521:2-aminobenzoic acid 2-(bromoacetyl)hydrazide; D001549:Benzamides; D005456:Fluorescent Dyes; C099522:N-(2-((bromoacetyl)amino)ethyl)-2-(methylamino)benzamide; D013439:Sulfhydryl Reagents; D062367:ortho-Aminobenzoates