Nanosecond Time-resolved Fluorescence Spectra of a Protein-Dye Complex

Brand, Ludwig; Gohlke, James R.

doi:10.1016/s0021-9258(19)77222-6

Cited by 113 publications

(19 citation statements)

References 9 publications

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“…Substituted aminonaphthalenesulfonate (ANS) chromophores have been widely used as fluorescent probes in proteins and membranes , due to the large variations in their fluorescence properties in environments of different polarity. For example, these chromophores exhibit strong fluorescence in apolar environments but weak fluorescence in water.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the time-dependent fluorescence of the chromophore is affected as the solvent reorganizes around the excited chromophore. In recent years, several groups have studied the time-resolved solvation of similar chromophores. ,, Dependent on the viscosity of the environment, the time scales on which these time-dependent solvent relaxation processes take place are on the order of a picosecond to several nanoseconds. ,, …”

Section: Resultsmentioning

confidence: 99%

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Fluorescent Probe Solubilization in the Headgroup and Core Regions of Micelles: Fluorescence Lifetime and Orientational Relaxation Measurements

1998

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Experimental results demonstrate that the fluorescent probes 2-(N-hexadecylamino)-naphthalene-6-sulfonate (HANS) and 2-(N-decylamino)-naphthalene-6-sulfonate (DANS) are solubilized in two distinct regions, that is, the headgroup and core, within micelles of cetyltrimethylammoniumbromide (CTAB), tetradecyltrimethylammoniumbromide (TTAB), dodecyltrimethylammoniumbromide (DTAB), cetyltrimethylammoniumchloride (CTAC), and tetradecyltrimethylammoniumchloride (TTAC). The fluorescence lifetime decays for both chromophores are biexponential in all the different micelles. The population associated with the shorter lifetime (τ 1 = 4-5 ns) is located in the Stern layer, where reduction of the fluorescence lifetime occurs because of quenching induced by the bromide counterions. The second population of chromophores is located in the hydrocarbon core region of the micelle. In this environment the chromophores have a considerably longer lifetime (τ 2 = 19-20 ns) because there is no significant quenching by bromide counterions. Evidence of water penetration places them fairly close to the core-Stern layer interface. Time-dependent fluorescence anisotropy is analyzed in terms of these two populations. The measurements show that the orientational relaxation of the probes in the hydrocarbon core region is considerably slower than orientational relaxation in the Stern layer. When the lifetime measurements and the orientational relaxation measurements are combined, the partitioning of the chromophores in the core and headgroup regions of the micelles can be determined.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Fluorescent Probe Solubilization in the Headgroup and Core Regions of Micelles: Fluorescence Lifetime and Orientational Relaxation Measurements

1998

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“…Nanosecond Spectrofluorometry. Time-resolved emission spectroscopy has been shown to be an important approach for obtaining direct kinetic evidence about processes occurring during the lifetime of the excited state (De Luca et al, 1971;Brand and Gohlke, 1971;Ware et at., 1968). We, therefore, used this technique to follow directly the relative rates of proton transfer from the excited-state phenol to solvent.…”

Section: Resultsmentioning

confidence: 99%

Excited-state proton transfer and the mechanism of action of firefly luciferase

Bowie¹,

Irwin²,

Loken³

et al. 1973

Biochemistry

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“…Solvent reorientation results in time-dependent spectral shifts. Such shifts have been observed for protein-and membrane-bound fluorophores [5,6,17,18,24], and these data can be used to estimate the dynamics of thk probe-binding site on the macromolecule. The data presented for TNS and PRODAN in viscous solvents illustrate the expected phase-modulation data for these and similar fluorophores when bound to macromolecules.…”

Section: Discussionmentioning

confidence: 97%

“…In the following sections we present similar measurements for the more complex case of solvent relaxation around the excited states of solvent-sensitive fluorophores. This process is an important determinant of the emission spectra of fluorophores bound to proteins and membranes [5,6,8,17,18].…”

Section: Spectral Relaxation In Viscous Solventsmentioning

confidence: 99%

Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Lakowicz

Balter

1982

Biophysical Chemistry

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Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-state protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (1) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (ø) larger than 90 degrees or by measurement of ratios of m/cos ø greater than 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overlap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and N-acetyl-L-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos ø measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.

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Nanosecond Time-resolved Fluorescence Spectra of a Protein-Dye Complex

Cited by 113 publications

References 9 publications

Fluorescent Probe Solubilization in the Headgroup and Core Regions of Micelles: Fluorescence Lifetime and Orientational Relaxation Measurements

Fluorescent Probe Solubilization in the Headgroup and Core Regions of Micelles: Fluorescence Lifetime and Orientational Relaxation Measurements

Excited-state proton transfer and the mechanism of action of firefly luciferase

Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

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