THE FLUORESCENCE PROPERTIES OF ORTHO AMINOBENZOATE ANESTHETICS IN DEFINED SOLVENTS AND PHOSPHOLIPID VESICLES

Abstract— The fluorescence properties of three ortho aminobenzoate local anesthetics have been determined in a variety of solvents. Results from these studies have been used to deduce how these drugs interact with phosphatidylcholine bilayers. The emission energy, fluorescence quantum yield and lifetime exhibited a biphasic dependence on solvent polarity. In aprotic solvents, alcohols and in ethanol-water mixtures containing less than 40% water, quantum yields and lifetimes were high (approximately 0.55 and 8.5 ns respectively). In ethanol-water mixtures containing >40% water, the strong fluorescence quenching was primarily due to an increase in the rate of non-radiative deactivation of the excited state. Both the radiative ( k_r ) and non-radiative ( k_nr ) rate constants show a biphasic dependence on solvent polarity. These studies suggest the presence of two singlet excited states for these molecules, a polar singlet excited state, S_1-p and a charge transfer excited state, S_1-ct with the latter predominating in ethanol-water mixture containing >40% water. In egg phosphatidylcholine bilayers, the fluorescence, lifetime and quantum yield are consistent with the view that these drugs are localized within the lipid head group region where the charge-transfer excited state can be stabilized by intermolecular hydrogen bonding.     

Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin

Fluorescence quenching and time-resolved fluorescence studies have been performed on the galactose-specific lectin purified from snake gourd (Trichosanthes anguina) seeds, in order to investigate the tryptophan accessibility and environment in the native protein and in the presence of bound ligand. Estimation of the tryptophan content by N-bromosuccinimide modification in the presence of 8 M urea yields four residues per dimeric molecule. The emission spectrum of native lectin in the absence as well as in the presence of 50 mM methyl--d-galatopyranoside (MeGal) shows a maximum around 331 nm, which shifts to 361.8 nm upon reduction of the disulfide bonds and denaturation with 8 M urea, indicating that all four tryptophan residues in the native state of this protein are in a hydrophobic environment. The extent of quenching that is observed is highest with acrylamide, intermediate with succinimide, and low with Cs⁺ and I⁻, further supporting the idea that the tryptophan residues are predominantly buried in the hydrophobic core of the protein. The presence of MeGal (50 mM) affects the quenching only marginally. Time-resolved fluorescence measurements yield bi-exponential decay curves with lifetimes of 1.45 and 4.99 ns in the absence of sugar, and 1.36 and 4.8 ns in its presence. These results suggest that the tryptophan residues are not directly involved in the saccharide binding activity of the T. anguina lectin. Of the four quenchers employed in this study, the cationic quencher, Cs⁺, is found to be a very sensitive probe for the tryptophan environment of this lectin and may be useful in investigating the environment of partially buried tryptophan residues and unfolding processes in other proteins as well.     

Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides

Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide c[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D- and 2D-(1)H NMR techniques. The peptide backbone has a single predominant conformation. The tryptophan side chain has three chi(1) rotamers: a major chi(1) = -60 degrees rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the chi(1) rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the chi(1) = -60 degrees rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to peptide bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8- and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the chi(1) = 180 degrees rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the epsilon-ammonium does not occur in these peptides.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

Synthesis and fluorescence characterization of MEHPPV oligomers

Trimer, tetramer, and pentamer oligomers based on the polymer backbone structure of poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] (MEHPPV) have been synthesized by Horner-Wadsworth-Emmons reactions. The fluorescence spectra, emission quantum yields, and lifetimes of the oligomers have been characterized in dilute chloroform solutions. The oligomers exhibit a sequential increase in absorption and emission wavelength maxima and a decrease in fluorescence lifetime as the π conjugation length is increased. The shortening in excited state lifetime is shown to be due to an increase in the rates of both radiative and nonradiative processes. The absence of a mirror-image relationship for the absorption and fluorescence spectra of the oligomers is attributed to the photoexcitation of a range of torsional configurations followed by relaxation to a more planar arrangement that then emits.     

THE FLUORESCENCE OF TRYPTOPHYL PEPTIDES

Abstract— Tryptophan lifetimes and relative quantum yields have been determined for a group of small (1–4 residues) peptides and peptide hormones [luteinizing releasing factor. mellitin, glucagon. glucagon 22–29, glucagon 1–26. glucagon 1–27 (homoserine)]. All of the larger peptides and most of the smaller peptide anions exhibit nonexponential decay. Peptide quenching in the small peptides is more effective when the bonding is at the amino rather than at the carboxyl end of tryptophan. With the exception of tryptophylglycine. quenching by NH⁺₃ is thought not to involve proton transfer. The results suggest that a decay component of 3–4 ns is expected whenever large peptides and proteins contain a solvent exposed tryptophan.     

The Correct Use of "Average" Fluorescence Parameters

When more than one fluorophore is present or when one fluorophore displays a multiple exponential decay "average" fluorescence parameters are derived, which can be combined with "average" lifetimes for further interpretation. However, two kinds of average lifetimes are used in this context: the intensity and the amplitude average lifetime. In this paper the different average parameters are carefully analyzed and their "best" combinations are derived. These average parameters are analyzed in the context of external and internal dynamic and static quenching, Föster energy transfer and the calculation of the radiative rate constant. The use of the amplitude average lifetime for the analysis of multiple fluorophore-containing systems and the detection of interactions is discussed.     

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.     

Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.     

HETEROGENEITY IN THE THERMALLY-INDUCED QUENCHING OF THE PHOSPHORESCENCE OF MULTI-TRYPTOPHAN PROTEINS

Abstract— The steady-state intensity and lifetime of the tryptophan phosphorescence from a number of globular proteins in 2:1 (v/v) glycerol buffer were monitored as a function of temperature. The phosphorescence lifetimes are essentially independent of the tryptophan local environment in rigid solution at temperatures < 170K, but vary markedly between proteins at temperatures at which the solutions become fluid. The ratio of steady-state intensity to lifetime P/τ was found to be temperature independent despite the quenching for free tryptophan and the lone residue in myelin basic protein. Heterogeneity in the triplet quenching of the tryptophans in liver alcohol dehydrogenase and alkaline phosphatase were revealed as step-like decreases in the ratio of P/T followed by plateau regions characterizing the homogeneous behavior of the more resistent tryptophans in the proteins. This heterogeneity exists not only between solvent-exposed and buried residues, but reflects variations in the flexibility of the structure surrounding distinct buried tryptophans in the globular proteins.     

Solvent effects on the fluorescence quenching of tryptophan by amides via electron transfer. Experimental and computational studies

Hybrid quantum mechanical/molecular mechanics (QM-MM) calculations [Callis and Liu, J. Phys. Chem. B 2004, 108, 4248-4259] make a strong case that the large variation in tryptophan (Trp) fluorescence yields in proteins is explained by ring-to-backbone amide electron transfer, as predicted decades ago. Quenching occurs in systems when the charge transfer (CT) state is brought below the fluorescing state (1L(a)) as a result of strong local electric fields. To further test this hypothesis, we have measured the fluorescence quantum yield in solvents of different polarity for the following systems: N-acetyl-L-tryptophanamide (NATA), an analogue for Trp in a protein; N-acetyl-L-tryptophan ethyl ester (NATE), wherein the Trp amide is replaced by an ester group, lowering the CT state energy; and 3-methylindole (3MI), a control wherein this quenching mechanism cannot take place. Experimental yields in water are 0.31, 0.13, and 0.057 for 3MI, NATA, and NATE, respectively, whereas, in the nonpolar aprotic solvent dioxane, all three have quantum yields near 0.35, indicating the absence of electron transfer. In alkyl alcohols the quantum yield for NATA and NATE is between that found for water and that found for dioxane, and it is surprisingly independent of chain length (varying from methanol to decanol), revealing that microscopic H-bonding, and not the bulk dielectric constant, dictates the electron transfer rate. QM-MM calculations indicate that, when averaged over the six rotamers, the greatly increased quenching found in water relative to dioxane can be attributed mainly to the larger fluctuations of the energy gap in water. These experiments and calculations are in complete accord with quenching by a solvent stabilized charge transfer from ring to amide state in proteins.     

Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.     

THE QUENCHING OF TYROSINE AND TRYPTOPHAN FLUORESCENCE BY H2O AND D2O*

Abstract— The quantum yields and lifetimes of the fluorescence of tyrosine and tryptophan were determined in D₂O-H₂O and glycerol-H₂O solvent mixtures of varying composition from 10 vol.% to 100% H₂O at 15°C. Forboth amino acids the ratio of the quantum yields in D₂O and H₂O (i.e., q_D/q_H) was smaller than the ratio of the corresponding lifetimes (_D/_H). For tyrosine the ratio of the quantum yields in glycerol and H₂O (q_G/q_H) was also smaller than the corresponding _G/_H ratio, but for tryptophan q_G/g_HG/_H. The proximity of the q vs. plots for tyrosine in the two solvent mixtures indicates that at 15°C neither D₂O nor glycerol, in the pure state or when diluted with H₂O, quench tyrosine significantly. However, H₂O quenches tyrosine by a dynamic process, which increases both the radiative and the nonradiative rate constant. The quenching action is attributed to a tyrosine-H₂O exciplex, whose formation is independent of bulk viscosity and dielectric constant. Unlike tyrosine, tryptophan is quenched weakly by D₂O by a static process at 15°C (i.e., involving no change in), but H₂O quenches tryptophan much more efficiently by a dynamic process, which involves the nonradiative rate constant, but not the radiative constant. These results are explained on the basis of electrostatic complexation of the ammonium group to the carbon atom adjacent to the ring nitrogen with a lifetime which is longer thanin D₂O but shorter thanin H₂O, with solvent reorientation possibly also being an important factor in the quenching. This explanation is consistent with the fact that concentrated (8 M) urea increases q andof aqueous tryptophan ? 15–20%, while guanidine hydrochloride (6.4 M) has the opposite effect, i.e., it decreases q and t of tryptophan ? 15–20%, and with the fact that neither 8 M urea nor 6.4 M guanidine hydrochloride affects any fluorescence parameter of tyrosine at all.     

Energy migration in novel pH-triggered self-assembled beta-sheet ribbons

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose-water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission.     

Effect of halogenated compounds on the photophysics of C70 and a monoadduct of C70: Some implications on optical limiting behaviour

The fluorescence spectra, quantum yields and lifetimes of C₇₀ and a pseudo-dihydro derivative (C₇₀R) have been measured in a wide range of solvents at room temperature. This information is important for the development of reverse saturable absorbers. Phosphorescence spectra and phosphorescence lifetimes were also measured at low temperature. The fluorescence is subject to quenching by halogenated compounds. The efficiency of quenching follows the order I>Br≫Cl. The nature of the quenching is shown to vary, with chlorinated compounds exhibiting static quenching of fullerene fluorescence, owing to nonfluorescent complex formation, whilst those compounds containing bromine and iodine exhibit dynamic quenching due to the external heavy-atom effect, that increases the intersystem crossing rate constant in the fluorophore–perturber complex. This constant is evaluated by an original method from the bimolecular quenching rate constants. The phosphorescence quantum yield of both fullerenes at 77 K slightly increases in the presence of iodobenzene, in spite of a strong decrease in phosphorescence lifetime. The marked increase of the intersystem crossing rate constant in concentrated solutions owing to the external heavy-atom effect is of interest for the application of fullerenes as fast-responding optical limiters (reverse saturable absorbers) of intense laser pulses, even in cases where the triplet quantum yield is of the order of unity.     

Fluorescence quenching of flavins by reductive agents

The fluorescence behaviour of the flavins riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and lumiflavin in aqueous solution at pH 8 in the presence of the reducing agents β-mercaptoethanol (β-ME), dithiothreitol (DTT), and sodium nitrite (NaNO₂) is studied under aerobic conditions. The fluorescence quantum yields and fluorescence lifetimes are determined as a function of the reducing agent concentration. For all three reducing agents diffusion controlled dynamic fluorescence quenching is observed which is thought to be due to photo-induced reductive electron transfer. For DTT additionally static fluorescence quenching occurs.     

1,3,5-三芳基-2-吡唑啉化合物光物理行为的研究

合成了一组带不同取代基的三芳基吡唑啉化合物,对它们在不同极性溶剂中的光物理行为(如荧光量子产率,荧光寿命等)进行了测定指出:这类化合物在光的激发下除存在有分子内共轭条件下的电荷转移行为外,还存在着分子内非共轭条件下的电子转移,本工作还以三芳基吡唑啉化合物为猝灭剂对氧鎓盐的荧光猝灭能力进行了研究,并对所得结果作了讨论。     

ROOM-TEMPERATURE PHOSPHORESCENCE FROM AZURIN DERIVATIVES. PHOSPHORESCENCE QUENCHING IN OXIDIZED NATIVE AZURIN

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.     

OPTICALLY DETECTED MAGNETIC RESONANCE OF THE TRYPTOPHAN PHOSPHORESCENT STATE IN NATIVE PROTEINS*

Abstract— The phosphorescent triplet state of tryptophan has been studied by the method of optically detected magnetic resonance (ODMR) at pumped helium temperatures in zero magnetic field. Only one of the triplet sublevels is found to be significantly radiative; the other two decay radiationlessly. Although the phosphorescence and ODMR decay lifetimes are influenced by spin–lattice relaxation processes at T = 1.3°K, the lifetime of the radiative level can be estimated as approximately 2 s, whereas the lifetimes of the non–radiative levels are in excess of 10 s. Comparison of the ODMR signals and the phosphorescence spectra has been made for tryptophans in native proteins with the following results: the ODMR signals of the two types of tryptophan sites in horse liver alcohol dehydrogenase can be resolved due to a shift in the D and E values of the respective triplet states; binding of the substrate tri- N -acetylglucosamine to hen lysozyme leads to a considerable narrowing of the phosphorescence peaks and ODMR signals as well as to a shift in the E value of the triplet state.
The following tentative conclusions can be reached: the tryptophan triplet D and E values are measurably affected by the environment of the chromophore in the protein, as are the linewidths of the magnetic resonance transitions. The | E | value is reduced and the magnetic resonance linewidth is increased with increasing exposure of the tryptophan to hydroxylic solvent. Although a considerable part of the width of the magnetic resonance transition can be ascribed to a heterogeneity of environments in the sample, there appears to exist an intrinsic line–broadening process which at present is not understood.