Analysis of two-state excited-state reactions.  The fluorescence decay of 2-naphthol

Laws, William R.; Brand, Ludwig

doi:10.1021/j100470a007

Cited by 219 publications

(170 citation statements)

References 9 publications

(14 reference statements)

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“…Nonetheless, it is known that the fluorescence lifetimes of probes can be altered by oxygen (33,34), pH (35,36), calcium (37,38), energy transfer (39)(40)(41), and a variety of other factors and/or quenchers (9,(42)(43)(44)(45)(46)(47). To date, most fluorescence sensors have not been characterized by lifetime measurements, and it is probable that such data will reveal additional probes that are useful for FLIM measurements.…”

Section: Experimental Methodsmentioning

confidence: 99%

Fluorescence lifetime imaging of free and protein-bound NADH.

Lakowicz

Szmacinski

Nowaczyk

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

669

582

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We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challengi case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetimeimages are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images, each obtained with various phase shifts of the ginmodulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe an imang procedure that allows speyific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.The phenomenon of fluorescence is widely utilized for research in the biosciences. These applications have been focused on two divergent types of measurements, timeresolved fluorescence and fluorescence microscopy. In the former, one takes advantage of the high information content ofthe time-dependent fluorescence decays to uncover details about the structure and dynamics of macromolecules (1-3). Such measurements are performed almost exclusively by using picosecond laser sources coupled with high-speed photodetectors. In contrast, fluorescence microscopy, in combination with dyes, stains, fluorophores, or fluorophorelabeled antibodies, is most often used to determine the localization of species of interest, usually proteins or other macromolecules (4-6). The acquisition of two-dimensional (2D) fluorescence images is typically accomplished with low-speed accumulating detectors. Consequently, the high information content of time-resolved fluorescence is not usually available for studies of microscopic biological samples. This is particularly disadvantageous when one considers the sensitivity of fluorescence decay times to chemical and environmental factors of interest, such as local pH, cation concentration, oxygen, and polarity, to name a few.In the present report we combine measurements of fluorescence lifetimes with 2D imaging to create images in which the lifetimes at each pixel are used to create contrast in the images. While there have been reports...

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Section: Experimental Methodsmentioning

confidence: 99%

Fluorescence lifetime imaging of free and protein-bound NADH.

Lakowicz

Szmacinski

Nowaczyk

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

669

582

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“…If energy transfer were to occur, the a value for the acceptor residue would be expected to be larger than the a value for the donor [24]. This is due to the fact that energy transfer results in a positive contribution to the a of the acceptor and a negative contribution to the CI of the donor.…”

Section: Enzyme Alonementioning

confidence: 98%

Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex

Wasylewski¹,

Eftink²

1987

European Journal of Biochemistry

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A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20°C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329-nm maximum and a 18.4-kJ/mol activation energy, E,, for thermal quenching. The longlifetime (3.5 ns) component has a 338-nm maximum and an E, of only 7.9 kJ/mol. Tentatively we assign the short and long-lifetime components to . Binding of the substrates ATP and 3-phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the E, for both components. Acrylamide-quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.Phosphoglycerate kinase (PGK) from yeast catalyzes the reversible phosphorylation of 3-phosphoglycerate by ATP in the glycolytic metabolic pathway [l]. X-ray and amino acid sequence studies [2 -41 have shown that the protein is a single polypeptide chain, containing 41 5 amino acid residues, and that it is folded into a bilobal structure. In this structure two domains of similar size are connected by a narrow 'waist' or 'hinge' region containing two helices [2, 51. The nucleotidebinding site is located on the C-terminal domain. The Nterminal domain contains the phosphoglycerate-binding site. Because these sites are separated by a distance of 1.2 -1.5 nm, a hinge-bending mechanism of the domain, which can bring the two substrates together for catalysis, has been proposed [6]. Solution studies have also shown that binding of substrates leads to conformational changes in the protein's structure [7 -121. Low-angle X-ray scattering [8] and ultracentrifugation [9] methods support this suggestion by showing that the binding of ATP and 3-phosphoglycerate to the enzyme reduces the radius of gyration [8] and increases the sedimentation coefficient [9] of the protein.Because a hinge-bending mechanism must involve rearrangement of the interdomain contact, it has been suggested that rotation of the two helices, which form the domain interface, occurs and, in doing so, brings the respective domains closer together [5].Tryptophan fluorescence has often been used as a sensitive probe of protein structure. PGK from yeast has only two tryptophan residues, both of which are located on the Cterminal domain [4]. One of the tryptophanyl residues, Trp-333, is located near the ATP-binding site. The second one, Using time-resolved fluorescence spectroscopy, Privat et al. [13] have shown that the tryptophanyl fluorescence of the enzyme can ...

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“…Although it is known that the presence of buffer species and their concentration may influence the rate of proton transfer, 29 studies of DHICA fluorescence kinetics in buffer solutions with different cations and anions did not provide conclusive evidence supporting this interpretation (A. Corani et al, unpublished). We were therefore prompted to take into account other possibilities for the observed spectral and dynamic properties of DHICA − .…”

mentioning

confidence: 99%

Excited-State Proton-Transfer Processes of DHICA Resolved: From Sub-Picoseconds to Nanoseconds

Corani

Pezzella

Pascher

et al. 2013

J. Phys. Chem. Lett.

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Excited-state proton transfer has been hypothesized as a mechanism for UV energy dissipation in eumelanin skin pigments. By using time-resolved fluorescence spectroscopy, we show that the previously proposed, but unresolved, excited-state intramolecular proton transfer (ESIPT) of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) occurs with a time constant of 300 fs in aqueous solution but completely stops in methanol. The previously disputed excited-state proton transfer involving the 5-or 6-OH groups of the DHICA anion is now found to occur from the 6-OH group to aqueous solvent with a rate constant of 4.0 × 10 8 s −1 .SECTION: Spectroscopy, Photochemistry, and Excited States T he 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a key product of tyrosine metabolism in cutaneous melanocytes, plays important roles in skin homeostasis as a major precursor with 5,6-dihydroxyindole (DHI) of eumelanin biopolymers, the dark pigments of human skin, hair, and eyes, 1 as an antioxidant, 2 and as a central mediator in cell−cell communication. 3 Eumelanins contain various proportions of DHICA-derived units ranging from only a few % up to >50% depending on the phenotype and organism. While eumelanins appear to be complex biopolymers in which the various units concur to determine the macroscopic properties at several levels of structural organization, redox states, and disorder, a detailed understanding of the photophysical behavior of key building blocks is crucial if structure−property−function relationships are to be drawn. The peculiar absorption features of eumelanin 4,5 and its former monomer DHI and DHICA might play a role in protecting the skin against UV radiation, though the UV-induced reaction mechanisms are largely unknown. Recent time-resolved spectroscopy work on DHICA 6−8 revealed a rich, pH-dependent photochemistry in aqueous buffer solution. At a pH where the carboxyl group of the molecule is fully protonated (e.g., pH 2.5), a red-shifted fluorescence band (λ max ≈ 430 nm) with a relatively short lifetime of 240 ps was observed and attributed to a zwitterionic species formed as a result of rapid excited-state intramolecular proton transfer (ESIPT) from the COOH group toward the NH group. 6 However, the actual transfer time and decay of the original excited state of the fully protonated molecule could not be resolved with the temporal resolution of the employed streak camera technique. By using femtosecond fluorescence upconversion (FU), we can now directly resolve this reaction step and show that it indeed proceeds on the sup-picosecond time scale.The deprotonated carboxylate anion DHICA − was observed to have a long (∼1.6 ns) lifetime in neutral (pH 7) buffer solution representing decay to a new species with a red-shifted fluorescence spectrum (λ max ≈ 450 nm) and a 2.4 ns lifetime. 6 This species was assigned to a complex between the excited DHICA − and a buffer species formed through a diffusion process. 6 On the other hand, calculations by Olsen et al. 9 suggested that...

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Analysis of two-state excited-state reactions. The fluorescence decay of 2-naphthol

Cited by 219 publications

References 9 publications

Fluorescence lifetime imaging of free and protein-bound NADH.

Fluorescence lifetime imaging of free and protein-bound NADH.

Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex

Excited-State Proton-Transfer Processes of DHICA Resolved: From Sub-Picoseconds to Nanoseconds

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