Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.
TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.
Lin
W Y
WY
Department of Chemistry, Pennsylvania State University, University Park 16802.
Eads
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CD
Villafranca
J J
JJ
D001667:Binding, Competitive; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D007811:Lanthanum / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D009282:Naphthalenesulfonates; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D008670:Metals; D009282:Naphthalenesulfonates; D013725:Terbium; D007811:Lanthanum; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D005974:Glutamate-Ammonia Ligase