Double-exponential Fluorescence Decay Research Articles

Temperature Dependence of Absorption and Energy Transfer Efficiency of Er3+/Yb3+/P5+ Co-Doped Silica Fiber Core Glasses.

A high phosphorus Er3+/Yb3+ co-doped silica (EYPS) fiber core glass was prepared using the sol-gel method combined with high-temperature sintering. The absorption spectra, emission spectra, and fluorescence decay curves were measured and compared in temperatures ranging from 300 to 480 K. Compared to 915 and 97x nm, the absorption cross-section at ~940 nm (~0.173 pm2) demonstrates a weaker temperature dependence. Hence, the 940 nm pump mechanism is favorable for achieving a high-power laser output at 1.5 μm. Additionally, the double-exponential fluorescence decay of Yb3+ ions and the emission intensity ratio of I1018nm/I1534nm were measured to evaluate the energy transfer efficiency from Yb3+ ions to Er3+ ions. Through the external heating and active quantum defect heating methods, the emission intensity ratios of I1018nm/I1534nm increase by 30.6% and 709.1%, respectively, from ~300 to ~480 K. The results indicate that the temperature rises significantly reduce the efficiency of the energy transfer from the Yb3+ to the Er3+ ions.

Interactions and Supramolecular Organization of Sulfonated Indigo and Thioindigo Dyes in Layered Hydroxide Hosts.

Supramolecularly organized host-guest systems have been synthesized by intercalating water-soluble forms of indigo (indigo carmine, IC) and thioindigo (thioindigo-5,5'-disulfonate, TIS) in zinc-aluminum-layered double hydroxides (LDHs) and zinc-layered hydroxide salts (LHSs) by coprecipitation routes. The colors of the isolated powders were dark blue for hybrids containing only IC, purplish blue or dark lilac for cointercalated samples containing both dyes, and ruby/wine for hybrids containing only TIS. The as-synthesized and thermally treated materials were characterized by Fourier transform infrared, Fourier transform Raman, and nuclear magnetic resonance spectroscopies, powder X-ray diffraction, scanning electron microscopy, and elemental and thermogravimetric analyses. The basal spacings found for IC-LDH, TIS-LDH, IC-LHS, and TIS-LHS materials were 21.9, 21.05, 18.95, and 21.00 Å, respectively, with intermediate spacings being observed for the cointercalated samples that either decreased (LDHs) or increased (LHSs) with increasing TIS content. UV-visible and fluorescence spectroscopies (steady-state and time-resolved) were used to probe the molecular distribution of the immobilized dyes. The presence of aggregates together with the monomer units is suggested for IC-LDH, whereas for TIS-LDH, IC-LHS, and TIS-LHS, the dyes are closer to the isolated situation. Accordingly, while emission from the powder H2TIS is strongly quenched, an increment in the emission of about 1 order of magnitude was observed for the TIS-LDH/LHS hybrids. Double-exponential fluorescence decays were obtained and associated with two monomer species interacting differently with cointercalated water molecules. The incorporation of both TIS and IC in the LDH and LHS hosts leads to an almost complete quenching of the fluorescence, pointing to a very efficient energy transfer process from (fluorescent) TIS to (nonfluorescent) IC.

Crystal structure, morphology and luminescent properties of rare earthion-doped SrHPO4 nanomaterials

Undoped and rare earth ions (Eu3+, Ce3+, Tb3+) doped β-SrHPO4 nanomaterials were successfully prepared by a facile hydrothermal method. The crystal structure, morphology and luminescent properties were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), photoluminescence (PL) spectra and luminescence decay curves. The results indicated that the undoped and rare earth ions doped SrHPO4 nanomaterials (the doping concentration was not above 7%) were well crystallized, with the same crystal structure (β-SrHPO4). Nevertheless, the doping of rare earth ions could lead to the changing of morphology from nanoflakes to nanocrystals. Under the excitation of UV light, rare earth ions (Eu3+, Ce3+, Tb3+) doped β-SrHPO4 nanocrystals exhibited the characteristic emission of Eu3+, Ce3+ and Tb3+ ions, respectively. The luminescence decay curves of β-SrHPO4:Eu3+ and β-SrHPO4:Ce3+,Tb3+ nanocrystals conformed to the double exponential fluorescence decay, and the average lifetimes were 1.14 and 4.12 ms, respectively. The luminescence decay curve of β-SrHPO4:Ce3+ was fitted into a single exponential function, and the lifetime was about 0.78 ns.

Synthesis and luminescence properties of blue-emitting phosphor K2Ca2Si2O7:Ce3+

A novel blue-emitting phosphor K2Ca2Si2O7:Ce3+ has been synthesized via a solid-state reaction. The pure phase structure of K2Ca2Si2O7:Ce3+ phosphor was investigated by using the XRD technique and Rietveld refinement. K2Ca1.98Si2O7:0.02Ce3+ phosphor exhibited broad-band blue emission peaking at 413nm upon 291nm excitation and 423nm upon 338nm excitation. The critical quenching concentration of Ce3+ was determined about 2mol% and the corresponding concentration quenching mechanism was considered to be the dipole–dipole interaction. The double-exponential fluorescence decay curve was also discussed to verify the two kinds of Ce3+ centre in K2Ca2Si2O7 host.

Picosecond Dynamics of Proton Transfer of a 7-Hydroxyflavylium Salt in Aqueous–Organic Solvent Mixtures

The intermediacy of the geminate base-proton pair (A*···H(+)) in excited-state proton-transfer (ESPT) reactions (two-step mechanism) has been investigated employing the synthetic flavylium salt 7-hydroxy-4-methyl-flavylium chloride (HMF). In aqueous solution, the ESPT mechanism involves solely the excited acid AH(+)* and base A* forms of HMF as indicated by the fluorescence spectra and double-exponential fluorescence decays (two species, two decay times). However, upon addition of either 1,4-dioxane or 1,2-propylene glycol, the decays become triple-exponential with a term consistent with the presence of the geminate base-proton pair A*···H(+). The geminate pair becomes detectable because of the increase in the recombination rate constant, k(rec), of (A*···H(+)) with increasing the mole fraction of added organic cosolvent. Because the two-step ESPT mechanism splits the intrinsic prototropic reaction rates (deprotonation of AH(+)*, k(d), and recombination, k(rec), of A*···H(+)) from the diffusion controlled rates (dissociation, k(diss), and formation, k(diff)[H(+)], of A*···H(+)), the experimental detection of the geminate pair provides a wealth of information on the proton-transfer reaction (k(d) and k(rec)) as well as on proton diffusion/migration (k(diss) and k(diff)).

ChemInform Abstract: Intramolecular Charge Transfer and Thermal Exciplex Dissociation with p-Aminobenzonitriles in Toluene.

The rate constants of the reversible intramolecular excited-state charge-transfer (CT) reaction of 4-(dimethylamino)benzonitrile (DMABN;I) in toluene were determined as a function of temperature, from double-exponential fluorescence decays measured by time-correlated single-photon counting. The same decay times were found for the locally excited (LE) and the CT state, as shown by global analysis. For the CT state, the stabilization enthalpy {Delta}H ({minus}6 kJ mol{sup {minus}1}) and the change in entropy {Delta}S ({minus}24 J K{sup {minus}1} M{sup {minus}1}) were determined. A correlation of the charge-transfer time of DMABN with the longitudinal dielectric relaxation time was not observed. The presence of dimers or solute/solvent complexes in the ground state was excluded by {sup 1}H NMR experiments. A reversible excited-state reaction was shown to occur in toluene for the planar molecules 1-methyl-5-cyanoindoline (NMCI; II) and 1-methyl-6-cyano-1,2,3,4-tetrahydroquinoline (NMCQ;III) as well as for 3,5-dimethyl-4-(dimethylamino)benzonitrile (DMADBN;IV), based on the wavelength dependence of the double-exponential fluorescence decays. Such a dependence was not found for N-methyl-4-aminobenzonitrile (MABN; IH), 4-aminobenzonitrile (ABN; IHH), and 3,5-dimethyl-4-aminobenzonitrile (ADBN; IVHH), which is attributed to an increase in energy of the CT state relative to the LE state in these secondary and primary aromatic amines.

Intramolecular charge transfer of 4-(dimethylamino)benzonitrile probed by time-resolved fluorescence and transient absorption: No evidence for two ICT states and a πσ∗ reaction intermediate

For the double exponential fluorescence decays of the locally excited (LE) and intramolecular charge transfer (ICT) states of 4-(dimethylamino)benzonitrile (DMABN) in acetonitrile (MeCN) the same times tau(1) and tau(2) are observed. This means that the reversible LE<==>ICT reaction, starting from the initially excited LE state, can be adequately described by a two state mechanism. The most important factor responsible for the sometimes experimentally observed differences in the nanosecond decay time, with tau(1)(LE)<tau(1)(ICT), is photoproduct formation. By employing a global analysis of the LE and ICT fluorescence response functions with a time resolution of 0.5 ps/channel in 1200 channels reliable kinetic and thermodynamic data can be obtained. The arguments presented in the literature in favor of a pisigma( *) state with a bent CN group as an intermediate in the ICT reaction of DMABN are discussed. From the appearance of an excited state absorption (ESA) band in the spectral region between 700 and 800 nm in MeCN for N,N-dimethylanilines with CN, Br, F, CF(3), and C(=O)OC(2)H(2) p-substituents, it is concluded that this ESA band cannot be attributed to a pisigma( *) state, as only the C-C[Triple Bond]N group can undergo the required 120 degrees bending.

Acceleration of Electron-Transfer-Induced Fluorescence Quenching upon Conversion to the Signaling State in the Blue-Light Receptor, TePixD, from Thermosynechococcus elongatus

TePixD is a blue light using flavin (BLUF) protein of a thermophilic cyanobacterium, Thermosynechococcus elongatus. The fluorescence dynamics of TePixD was observed for the first time in both its dark-adapted and signaling (red-shifted) forms with a 200-fs time resolution. The fluorescence up-conversion setup was used in the time region up to 60 ps, and the streak-camera setup was used in the time region up to 1 ns. To avoid the accumulation of the red-shifted form by the exciting laser irradiation, the sample solution was circulated using a diaphragm pump. A handmade flow cuvette with a small cross section was used to achieve a fast flow of the solution in the excited region. The fluorescence decay times were unequivocally determined to be 13.6 and 114 ps for the dark-adapted form and 1.37 ps for the red-shifted form. The double-exponential fluorescence decay in the dark-adapted form suggested the coexistence of two conformations that have the 13.6- and 114-ps decay components, respectively. The single-exponential fluorescence decay in the red-shifted form suggested the elimination of heterogeneity in the conformation upon the light-induced conversion. The fast fluorescence-quenching components were almost eliminated in the mutant in which the conserved tyrosine Tyr8 is replaced by phenylalanine. Thus, the fluorescence quench was concluded to arise from the electron transfer from Tyr8, to the excited flavin chromophore. The 10-fold-faster quenching in the red-shifted form suggested the acceleration of the electron transfer. The faster decay time of 13.6 ps for the dark-adapted form was found to be almost temperature independent in the region from 10 to 40 degrees C. This suggested that the energy gap, DeltaG, in Marcus's electron-transfer theory is optimized to give the fastest rate. The acceleration of the electron transfer in the red-shifted form is interpreted to be due to the enhancement of the electronic-coupling factor between the donor and acceptor. A shortening of the Tyr8-flavin distance by 1.0-1.5 A was suggested if we adopt the empirical formula for the donor-acceptor distance dependence of the electron transfer rate.

Protochlorophyll complexes with similar steady-state fluorescence characteristics can differ in fluorescence lifetimes. A model study in Triton X-100

The steady-state and time-resolved fluorescence characteristics of protochlorophyll (Pchl) dissolved in neat Triton X-100 and in Triton X-100 micelles were investigated, and the fluorescence lifetimes of different Pchl spectral forms were studied. Varying the concentration of Pchl or diluting the micellar solutions either with a buffer or with a micellar solution, 631–634, 645–655, 680–692 and above 700 nm emitting Pchl complexes were prepared, the ratios of which varied from one another. The fluorescence decay of the 631–634 nm emitting (monomeric) form had a mono-exponential character with a 5.4-ns fluorescence lifetime. The long-wavelength Pchl complexes (aggregates) had two fluorescence lifetime values within a range of 1.4–3.9 ns and 0.15–0.84 ns, which showed high variability in different environments. Depending on the conditions, either mono- or double-exponential fluorescence decay was found for a fluorescence band at 680–685 nm. These data show that despite their very similar steady-state fluorescence properties, Pchl complexes can differ in fluorescence lifetimes, which may reflect different molecular structures, intrinsic geometries or different molecular interactions. This underlines the importance of complex spectroscopic analysis for a precise description of native and artificial chlorophyllous pigment forms.

Dynamics of Ultrafast Intramolecular Charge Transfer with 4-(Dimethylamino)benzonitrile in Acetonitrile

The kinetics of the intramolecular charge-transfer (ICT) reaction of 4-(dimethylamino)benzonitrile (DMABN) in the polar solvent acetonitrile (MeCN) is investigated by fluorescence quantum yield and picosecond time-correlated single photon counting (SPC) experiments over the temperature range from -45 to +75 degrees C, together with femtosecond Sn <-- S1 transient absorption measurements at room temperature. For DMABN in MeCN, the fluorescence from the locally excited (LE) state is strongly quenched, with an unquenched to quenched fluorescence quantum yield ratio of 290 at 25 degrees C. Under these conditions, even very small amounts of the photoproduct 4-(methylamino)benzonitrile (MABN) severely interfere, as the LE fluorescence of MABN is in the same spectral range as that of DMABN. The influence of photoproduct formation could be overcome by a simultaneous analysis of the picosecond and photostationary measurements, resulting in data for the activation barriers Ea (5 kJ/mol) and Ed (32 kJ/mol) of the forward and backward ICT reaction as well as the ICT reaction enthalpy and entropy: DeltaH (-27 kJ/mol) and DeltaS [-38 J/(mol K)]. The reaction hence takes place over a barrier, with double-exponential fluorescence decays, as to be expected in a two-state reaction. From femtosecond transient absorption down to 200 fs, the LE and ICT excited state absorption (ESA) spectra of DMABN in n-hexane (LE) and in MeCN (LE and ICT) and also of 4-aminobenzonitrile in MeCN (LE) are obtained. For DMABN in MeCN, the quenching of the LE and the rise of the ICT ESA bands occurs with a single characteristic time of 4.1 ps, the same as the ICT reaction time found from the picosecond SPC experiments at 25 degrees C. The sharp ICT peak at 320 nm does not change its spectral position after a pump-probe delay time of 200 fs, which suggests that large amplitude motions do not take place after this time. The increase with time in signal intensity observed for the LE spectrum of DMABN in n-hexane between 730 and 770 nm, is attributed to solvent cooling of the excess excitation energy and not to an inverse ICT --> LE reaction, as reported in the literature.

Fluorescence Properties of Recombinant Tropomyosin Containing Tryptophan, 5‐Hydroxytryptophan and 7‐Azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.

Whole-field fluorescence lifetime imaging with picosecond resolution using ultrafast 10-kHz solid-state amplifier technology

We report the development of a high temporal resolution whole-field fluorescence lifetime imaging system based on an ultrafast solid-state laser system and a time-gated image intensifier operating at up to 10 kHz. The temporal instrument response is /spl sim/110 ps and we have imaged (environmentally perturbed) differences in fluorescence lifetime as small as 20 ps. Fluorophores exhibiting single- or double-exponential fluorescence decay profiles are routinely imaged and a near real-time update time of 3 s for the fluorescence lifetime map has been demonstrated using a modest personal computer. We also present provisional fluorescence lifetime images of tissue constituents. This fluorescence lifetime imaging technology is applicable to almost any optical instrument configuration and, when coupled with existing all-solid-state diode-pumped ultrafast laser technology, may yield a potentially inexpensive instrument for in vitro and in vivo biomedical imaging.

Analysis of double exponential fluorescence decay behavior for optical temperature sensing

Algorithms based on Prony’s method are proposed for the estimation of exponential time constants of a double exponential fluorescence–decay process. The performance of these algorithms is evaluated, using Monte Carlo simulations, the results of which are presented showing that the performance of Prony’s method can be greatly improved with the selection of an optimized observation window width and a few algorithm-related parameters. The applications of these noniterative algorithms in real-time, quasidistributed temperature sensor systems are discussed where the material decay may not approximate to a single exponential, or multimaterial sensors are used with a single, simple pump source and a single detector. Applications to quasidistributed sensor systems are considered and, thus, experimental results are presented to justify the use of the algorithms in Prony’s method in practical double exponential fluorescence decay sensor analysis.

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human α-lactalbumin (α-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins. The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules. Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and α-HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.

Time-resolved microfluorometric study of the binding sites of lipophilic cationic pyrene probes in mitochondria of living HeLa cells

Lipophilic dye cations specifically bind to the mitochondria of living cells. Using fluorescent dyes, the mitochondria can easily be observed with a fluorescence microscope. Electron microscopy has shown that the dyes are bound to the inner mitochondrial membranes and the cristae. Using time-resolved fluorescence microscopy we have investigated, whether the dye molecules are preferentially accumulated at the strongly hydrophobic protein complexes of energy metabolism or at the lipids of the inner membrane system. In order to use our nanosecond-pulsed laser fluorometer we synthesized specially designed lipophilic pyrene cations with S 1 lifetimes in the nanosecond domain, which specifically stain mitochondria in living HeLa cells. Model experiments with artificial membranes such as liposomes, proteoliposomes and also protein complexes have shown that the fluorescence is strongly quenched by oxygen if the pyrene probes are bound to lipids. Binding to proteins causes a much smaller quenching effect. In artificial systems, all decays were single exponential. This is in contrast with incubated HeLa cells, which showed double-exponential fluorescence decays. Comparing these with the artificial systems we came to the conclusion that in HeLa cells the long-lived species 1 are pyrene probes preferentially bound to the proteins of the inner mitochondrial membranes. The short-lived species 2 is caused by fluorescence resonance energy transfer from the pyrene probes as donors to cytochromes of the inner membranes as acceptors. From our decay data we estimated a mean distance between donor and acceptor of about 40 Å. This is the same order of magnitude as the mean diameters of several mitochondrial protein complexes. Therefore we assumed that species 2 are pyrene probes bound either to mitochondrial proteins with cytochromes as constituents or to the interface between these proteins and the phospholipids of the membranes. Thus both species 1 and species 2 are spatially related to mitochondrial proteins. This agrees with the observation that respiration of HeLa cells as well as cytochrome c oxidase vesicles (COVc) are inhibited with increasing concentration of pyrene probes. Finally, we studied the photodynamic effect on irradiation of HeLa cells and of COVs after incubation with lipophilic pyrene and porphyrine cations.

Intramolecular charge transfer and thermal exciplex dissociation with p-aminobenzonitriles in toluene

The rate constants of the reversible intramolecular excited-state charge-transfer (CT) reaction of 4-(dimethylamino)benzonitrile (DMABN;I) in toluene were determined as a function of temperature, from double-exponential fluorescence decays measured by time-correlated single-photon counting. The same decay times were found for the locally excited (LE) and the CT state, as shown by global analysis. For the CT state, the stabilization enthalpy {Delta}H ({minus}6 kJ mol{sup {minus}1}) and the change in entropy {Delta}S ({minus}24 J K{sup {minus}1} M{sup {minus}1}) were determined. A correlation of the charge-transfer time of DMABN with the longitudinal dielectric relaxation time was not observed. The presence of dimers or solute/solvent complexes in the ground state was excluded by {sup 1}H NMR experiments. A reversible excited-state reaction was shown to occur in toluene for the planar molecules 1-methyl-5-cyanoindoline (NMCI; II) and 1-methyl-6-cyano-1,2,3,4-tetrahydroquinoline (NMCQ;III) as well as for 3,5-dimethyl-4-(dimethylamino)benzonitrile (DMADBN;IV), based on the wavelength dependence of the double-exponential fluorescence decays. Such a dependence was not found for N-methyl-4-aminobenzonitrile (MABN; IH), 4-aminobenzonitrile (ABN; IHH), and 3,5-dimethyl-4-aminobenzonitrile (ADBN; IVHH), which is attributed to an increase in energy of the CT state relative to the LE state in these secondary and primary aromatic amines.

Lifetimes and collisional self-quenching rates for C 2H 2Ã 1A u ( v′ 3 = 0 to 4)

Time-resolved laser-induced fluorescence is used to monitor the population in excited rovibronic levels of the à 1A u state of C 2H 2 in v′ 3 = 0 to 4. Double-exponential fluorescence decays are observed in all excitation lines. From the pressure-dependent decay rates, zero-pressure lifetimes and self-quenching rates are derived.

Pressure effect on the fluorescence decay of erythrosin bonded to an aminopropyl silica gel particle — measurements under a microscope

An experimental technique to examine the effect of pressure on the fluorescence decay of a microscopic sample has been developed and applied to erythrosin (ER) chemically bonded to an aminopropyl silica gel particle (APS). A dye-bonded particle (ER  APS) of 3 – 10 μm in diameter exhibits a double-exponential fluorescence decay in all solvents studied; this reflects the presence of at least two environmentally different states of the ER moiety. A significant effect of pressure observed in ethylene glycol is ascribed to the pressure-assisted penetration of the solvent molecules into small pores in ER  APS. However, in benzene, ethanol and acetonitrile, no effect of pressure is observed on the fluorescence decay of ER  APS up to 50 MPa despite marked differences in the fluorescence decay in these solvents. This indicates that the two states responsible for the dual exponentiality and their interconversion are not influenced by increasing pressure.

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10°C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl 2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the proten. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, k q values, of 2.3 and 0.26·10 9 M −1·s −1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, E a values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca 2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca 2+ results in an increase in the exposure and the polarity of the microenvironment of this ‘blue’ residue.

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding.

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.