Energy transfer to a proton-transfer fluorescence probe: tryptophan to a flavonol in human serum albumin.
A protein fluorescence probe system, coupling excited-state intermolecular Förster energy transfer and intramolecular proton transfer (PT), is presented. As an energy donor for this system, we used tryptophan, which transfers its excitation energy to 3-hydroxyflavone (3-HF) as a flavonol prototype, an acceptor exhibiting excited-state intramolecular PT. We demonstrate such a coupling in human serum albumin-3-HF complexes, excited via the single intrinsic tryptophan (Trp-214). Besides the PT tautomer fluorescence (lambda max = 526 nm), these protein-probe complexes exhibit a 3-HF anion emission (lambda max = 500 nm). Analysis of spectroscopic data leads to the conclusion that two binding sites are involved in the human serum albumin-3-HF interaction. The 3-HF molecule bound in the higher affinity binding site, located in the IIIA subdomain, has the association constant (k1) of 7.2 x 10(5) M-1 and predominantly exists as an anion. The lower affinity site (k2 = 2.5 x 10(5) M-1), situated in the IIA subdomain, is occupied by the neutral form of 3-HF (normal tautomer). Since Trp-214 is situated in the immediate vicinity of the 3-HF normal tautomer bound in the IIA subdomain, the intermolecular energy transfer for this donor/ acceptor pair has a 100% efficiency and is followed by the PT tautomer fluorescence. Intermolecular energy transfer from the Trp-214 to the 3-HF anion bound in the IIIA subdomain is less efficient and has the rate of 1.61 x 10(8) s-1 thus giving for the donor/acceptor distance a value of 25.5 A.
Institute of Molecular Biophysics, Florida State University, Tallahassee 32306-3015, USA.
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Litvinyuk
D001665:Binding Sites; D004735:Energy Transfer; D005419:Flavonoids; D005456:Fluorescent Dyes; D006801:Humans; D008962:Models, Theoretical; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan
D005419:Flavonoids; D005456:Fluorescent Dyes; D012709:Serum Albumin; D014364:Tryptophan; C041477:3-hydroxyflavone