Phosphorescence quenching as an approach for estimating the localization of triplet label in cotton fibers

The method based on quantitative analysis of chromophore fluorescence (phosphorescence) quenching, for instance, by a stable nitroxide radical, was the first time used to measure the depth of immersion of a triplet label in cotton fiber as a molecular object. Erythrosine triplet labels were incorporated in cotton fibers with subsequent measurement of the efficiency of label phosphorescence quenching and of the temperature dependence of phosphorescence duration. Using the concept of dynamic quenching in solution and the empirical dependence of the parameters of static quenching between centers with fixed distance, we could estimate the depth of chromophore immersion in the fiber (7.4–7.8 dynamics of the label in the millisecond range of correlation times. Subtle differences in microstructure and molecular dynamics were revealed between fiber specimens from healthy and diseased cotton. The proposed approach can be used for investigating a wide scope of biological and nonbiological objects.     

Methods of physical labels--a combined approach to the study of microstructure and dynamics in biological systems

The physical principles of several new approaches to the investigation of biological and model systems are discussed, including versions of the spin label method based on relaxation measurements, and also the methods of triplet, M?ssbauer, electron-scattering and radical-pair labels and probes. It is shown that all these methods make it possible to investigate molecular mobility of the medium with the correlation frequencies tau c-1 = 10(-3) -10(11) s-1, to measure the rate constants of collisions Ktr = 10(3) -10(10) M-1 s-1, to measure the distance between centers up to 100 A and finally, to evaluate the immersion depths of paramagnetic and chromophore centers in matrices up to 40 A. The combined approach is demonstrated with examples from studies of the structure of nitrogenase, the reaction centers of photosynthetic bacteria and sarcoplasmic reticulum membranes and from studies of the molecular dynamics of proteins and membranes.     

Structure and intramolecular dynamics of a photoactive pigment-protein eosin-casein complex

The structure of eosin--casein complex was studied by triplet label method. Quantitative data on the quantum--mechanic exchange interaction between eosin centres and external quenchers were obtained. The dynamic state of water--protein matrix at -20 degrees to -180 degrees C with eosin as fluorescence and phosphorescence labels and natural chromophores of protein--tryptophane was studied.     

Depth of immersion of fluorescent chromophores in biomembranes studied by quenching with nitroxide radical

A novel method has been developed for measuring depth of immersion of a fluorescent chromophore in biological matrices, such as biomembranes. The method is based on dynamic quenching of chromophore fluorescence by a nitroxide probe freely diffused in solution. Theoretical considerations and experimental evidences relating to the method are discussed. The proposed method was applied to investigation of lecithin liposomes and membranes from Bacillus subtilis modified by the photochrome-fluorescent probe 4,4'-dimethylaminocyanostilbene.     

Temperature dependence of the disulfide perturbation to the triplet state of tryptophan

Variability in the temperature dependence of disulfide quenching of the tryptophan phosphorescence of globular proteins in rigid glasses is illustrated with lysozyme and α-bungarotoxin. A laser-pulsed phosphorescence study of this short-range interaction with a model indole-disulfide system is described. The perturbation of secondary dibutyl disulfide on the triplet state of the indole moiety in 2-(3-indolyl)ethyl phenyl ketone in rigid media is found to display a bimodal temperature dependence. The quenching rate constant at contact between chromophore and perturber is observed to be temperature independent below 30 K, but to increase with temperature between 30 and 100 K with an activation energy of ~200 cm^-1. The results suggest that the underlying quenching interaction involves a photo-induced one-electron transfer from the excited state of indole to the disulfide.     

Quenching of triplet state fluorophores for studying diffusion-mediated reactions in lipid membranes

An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane-associated electron spin resonance labels on the triplet state kinetics of membrane-bound fluorophores. Singlet-triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied, when free in aqueous solutions, with LRB bound to a lipid in a liposome, and in the presence of different local concentrations of the electron spin resonance label TEMPO. By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy, a strong fluorescence signal can be combined with the ability to monitor low-frequency molecular interactions, at timescales much longer than the fluorescence lifetimes. Both in solution and in membranes, the measured relative changes in the singlet-triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules. These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected. The proposed strategy is broadly applicable, in terms of possible read-out means, types of molecular interactions that can be followed, and in what environments these interactions can be measured.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.     

Quenching of alkaline phosphatase phosphorescence by O2 and NO. Evidence for inflexible regions of protein structure.

The rate constant for quenching the phosphorescence of alkaline phosphatase by molecular oxygen was measured as a function of temperature. The results disagree with previous determinations and, contrary to fluorescence quenching, show that diffusion of O2 to this region of the macromolecule is a highly hindered process. When nitric oxide is introduced as a quencher, similarly small rate constants were found. While the activation energy for this process is identical for both quenchers, it is much smaller than for structural fluctuations at the chromophore site as manifested by the intrinsic triplet-state lifetime. These findings are analyzed in terms of a mechanism that takes into account static quenching at large distances and does not require penetration of the quencher all the way to the chromophore.     

Location of lysine-beta 162 in mitochondrial F1-adenosinetriphosphatase

The quenching of the fluorescence of bovine heart F1-adenosinetriphosphatase labeled specifically at its essential Lys-beta 162 with 7-chloro-4-nitro-2,1,3-benzoxadiazole (N-NBD-F1) by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-triphosphate (TNP-ATP) has been studied. Analysis of the fluorescence data in the presence of 1 mM ATP shows that the dissociation constant of TNP-ATP from its first binding site in the covalently labeled enzyme is 250-fold lower than that of ATP, which it replaces in pH 7.0 buffer containing 25% glycerol, and that this binding causes a 54% quenching of the fluorescence of the N-NBD label due to energy transfer to the weakly fluorescent TNP-ATP molecule. Computation based on the observed quenching gives a distance of 25.6 +/- 0.4 A between the NBD label and the chromophore of the bound TNP-ATP molecule. Since the distance between the chromophore and the farthest O atom of the bound TNP-ATP is about 16 A, it seems quite likely that the epsilon-amino group of Lys-beta 162 is near the gamma-phosphate group of the TNP-ATP bound at the catalytic site. Similar measurements in the presence of 1 mM ADP show that the replacement of ADP at the catalytic site by TNP-ATP causes a 49% quenching of the fluorescence of the N-NBD label, which gives a distance of 26.5 +/- 0.4 A between the label and the chromophore of the bound TNP-ATP molecule.     

Spectroscopic studies of arsenic(III) binding to Escherichia coli RI methyltransferase and to two mutants, C223S and W183F.

The interactions of an arsenic (III) reagent, (CH3)2AsSCH2CONH2, with two Escherichia coli RI methyltransferase mutants, W183F and C223S, have been studied by phosphorescence, optically detected magnetic resonance, and fluorescence spectroscopy. The phosphorescence spectrum of the W183F mutant containing only one tryptophan at position 225 reveals a single 0,0-band that is red-shifted by 9.8 nm upon binding of As(III). Fluorescence titration of W183F with (CH3)2AsSCH2CONH2 produces a large tryptophan fluorescence quenching. Analysis of the quenching data points to a single high-affinity As(III) binding site that is associated with the fluorescence quenching. Triplet-state kinetic measurements performed on the perturbed tryptophan show large reductions in the lifetimes of the triplet sublevels, especially that of the T chi sublevel. As(III) binding to the enzyme at a site very close to the Trp225 residue induces an external heavy-atom effect, showing that the perturber atom is in van der Waals contact with the indole chromophore. In the case of the C223S mutant, a single tryptophan 0,0-band also is observed in the phosphorescence spectrum, but no change occurs upon addition of the As(III) reagent. Fluorescence titration of C223S with As(III) shows essentially no quenching of tryptophan fluorescence, in contrast with W183F. These results, along with previous triplet-state and biochemical studies on the wild-type enzyme [Tsao, D. H.H., & Maki, A. H. (1991) Biochemistry 30, 4565-4572], show that As(III) binds with high affinity to the Cys223 residue and that the Trp225 side chain is located close enough to that of Cys223 to produce a heavy-atom perturbation when As(III) is bound.     

Distance dependence of the tryptophan-disulfide interaction at the triplet level from pulsed phosphorescence studies on a model system.

In the present study the distance dependence of tryptophan-disulfide interaction is examined with a view to both utilizing the interaction as a more quantitative indicator of subtle conformational changes in proteins as well as elucidating the interaction mechanism. To examine perturbations specifically at the indole triplet level 2-(3-indolyl)-ethyl phenyl ketone (IEPK) in which excitation is transferred with high efficiency to the triplet state of the indole moiety was employed. Phosphorescence decays of IEPK excited by a laser pulse in 70/30 (vol/vol) ethanolether at 77 K were measured in the presence of various concentrations of simple disulfides. The nonexponential phosphorescence decays arising from a distribution of fixed chromophoreperturber separations and the steady-state quenching of IEPK were accounted for with an exponential dependence of the quenching rate constant with distance. The small effective Bohr radius (0.8 A) that appears in the exponent emphasizes the localized nature of the interaction. Comparison of the triplet quenching rate constant obtained at quencher contact with IEPK to that estimated in proteins suggests a dependence on the triplet energy of the indole moiety and an endothermic nature for the quenching process. The study predicts that in proteins tryptophan-disulfide interactions are very localized in nature and should give rise to detectable anomalous decays only out to 2 A beyond van der Waals contact between the interacting partners.     

Study of kinetic parameters of singlet molecular oxygen in aqueous porphyrin solutions. Effect of detergents and the quencher sodium azide

The kinetic parameters of porphyrin-photosensitized formation and deactivation of singlet molecular oxygen (1O2) and their dependence on the concentration of the 1O2 quencher sodium azide were investigated in air-saturated water, ethanol, and aqueous micellar solutions of detergents using time-resolved measurements of oxygen phosphorescence under pulsed laser excitation. The lifetimes of 1O2 formation and deactivation and the rate constants of 1O2 quenching by sodium azide were determined. It was shown that, with no azide in the solutions, the rise in phosphorescence intensity after the laser flash corresponded to the kinetics of energy transfer from the porphyrin triplet molecules to oxygen, while the decay kinetics corresponded to the kinetics of 1O2 deactivation. In the presence of detergent, a considerable increase in the 1O2 lifetime was observed, which is likely due to the localization of 1O2 molecules mostly in lipophilic micelles and not in the water phase. If relatively high azide concentrations were used, the lifetime of the porphyrin triplet state did not change but the 1O2 lifetime decreased to values similar to those in living cells. In this case, the inversion of the phosphorescence kinetic phases was observed. The rise corresponded to 1O2 deactivation, and the decay, to the energy transfer from triplet porphyrin to oxygen. The data suggest that, in living cells, 1O2 molecules are also located mainly in lipophilic structures and the 1O2 lifetime determines the kinetics of the phosphorescence rise after the laser pulse.     

Dynamic site heterogeneity in amorphous maltose and maltitol from spectral heterogeneity in erythrosin B phosphorescence

We have used phosphorescence from erythrosin B (tetraiodofluorescein) dispersed in thin films of either maltose or maltitol to investigate the physical properties of these amorphous pure sugar matrixes. Intensity decays collected as a function of emission wavelength over the range from 640 to 720 nm were analyzed using a stretched exponential kinetic model in which the lifetime (tau) and the stretching exponent (beta) were the physically relevant parameters. The lifetimes varied systematically with emission wavelength in both matrixes. Analysis of the temperature dependence of the lifetime at each wavelength provided an estimate of the activation energy for nonradiative quenching of the triplet state; the activation energy also varied with emission wavelength. In addition, time-resolved emission spectra exhibited a blue shift with time following excitation. These data support a photophysical model in which probes are distributed among sites that vary in terms of overall molecular mobility and in which sites with lower rates of dipolar relaxation also have lower rates of collisional quenching of the erythrosin triplet state. The amorphous matrix of both maltose and maltitol in both the glass and the melt state is thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.     

Tryptophan phosphorescence of G-actin and F-actin

The tryptophan phosphorescence spectrum, intensity and decay kinetics of G-actin and F-actin were measured over a temperature range of 140-293 K. The fine structure in the phosphorescence spectra at low temperature, with O,O vibrational bands centered at 405 nm and 415.5 nm for both species, reveals a marked heterogeneity of the chromophore environment. The thermal quenching profile distinguishes these sites in terms of their flexibility, and shows that probably only one of the four tryptophan residues is still phosphorescent at ambient temperature due to its location in a relatively rigid buried core. Although some differences are demonstrated between G-actin and F-actin at low temperature, the identity of the triplet lifetime at ambient temperature strongly supports the notion that the conformation of the macromolecule is largely unaffected by polymerization. Preliminary phosphorescence anisotropy measurements demonstrate both the occurrence of singlet-singlet energy transfer among tryptophan residues and a strong immobilization of actin in the polymerized state.     

Molecular mobility and dynamic site heterogeneity in amorphous lactose and lactitol from erythrosin B phosphorescence

We have used phosphorescence from erythrosin B to characterize the molecular mobility and dynamic heterogeneity in dry films of amorphous lactose and lactitol from -25 to 120 degrees C. The phosphorescence emission spectra red-shifted and broadened with temperature in both sugars, indicating that both the rate of dipolar relaxation and the extent of inhomogeneous broadening increased dramatically at higher temperature. Phosphorescence intensity decays were well fit using a stretched exponential decay model; the rate constant for non-radiative quenching due to collisions with the matrix was calculated from the lifetimes. Arrhenius plots of this rate were non-linear, increasing very gradually at low and dramatically at high temperatures in both sugars. The rate of quenching was significantly lower in a 1:1 (wt/wt) mixture of lactose/lactitol in both the glass and the melt, providing strong evidence that specific interactions within the mixture lowered the matrix mobility. The lifetimes varied systematically with emission wavelength in both matrixes; analysis of the temperature dependence indicated that the activation energy for non-radiative quenching of the triplet state varied somewhat with emission wavelength. Time-resolved emission spectra collected as a function of delay time following pulsed excitation exhibited significant shifts to higher energy as a function of time. These data support a photophysical model in which erythrosin B molecules are distributed among matrix sites that vary such that blue-emitting sites with slower rates of matrix dipolar relaxation also have slower rates of molecular collisions. The amorphous matrixes of lactose and lactitol in both the glass and the melt state are thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin‐I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels.

Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues, such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching, the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).