title: Synthesis and spectral characterization of sulfhydryl-reactive fluorescent probes.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Pouchnik
authors.forename: L E
authors.initials: LE
authors.lastname: Laverman
authors.forename: F
authors.initials: F
authors.lastname: Janiak-Spens
authors.forename: D M
authors.initials: DM
authors.lastname: Jameson
authors.forename: G D
authors.initials: GD
authors.lastname: Reinhart
authors.forename: C R
authors.initials: CR
authors.lastname: Cremo
mesh_terms: 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
chemical_list: 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