Indole, fluorescence spectrum

The steady state spectra of indole solvated in water show similar behavior as those from indole solvated in ethanol (see Fig. la)). Due to the larger dipole moment of the water molecules a somewhat larger Stokes shift of the fluorescence spectrum is observed. 

From the steady state fluorescence spectrum of indole in water a fluorescence quantum yield of about 0.09 is determined. Since the cation appears in less than 80 fs a branching of the excited state population has to occur immediately after photo excitation. We propose the model shown in Fig. 3a). A fraction of 45 % experiences photoionization, whereas the rest of the population relaxes to a fluorescing state, which can not ionize any more. A charge transfer to solvent state (CITS), that was also introduced by other authors [4,7], is created within 80 fs. The presolvated electrons, also known as wet or hot electrons, form solvated electrons with a time constant of 350 fs. Afterwards the solvated electrons show no recombination within the next 160 ps contrary to solvated electrons in pure water as is shown in Fig. 3b). 

Figure 23-13 (A) Corrected emission and excitation spectra of riboflavin tetrabutyrate in w-heptane. Concentration, about 0.4 mg I-1. Curve 1 excitation spectrum emission at 525 nm. Curve 2 emission spectrum excitation at 345 nm. FromKotaki and Yagi.128 (B) Indole in cyclohexane, T = 196 K. 1, Fluorescence excitation spectrum 2, fluorescence spectrum and 3, phosphorescence spectrum. From Konev.125...

A fluorescence spectrum is characteristic of a given compound. It is observed as a result of radiative emission of the energy absorbed by the molecule. The observed spectrum does not depend on the wavelength of the exciting light, except that the spectrum will be more intense if irradiation occurs at the absorption maximum. The spectral intensity is called the quantum efficiency and is usually abbreviated as . The quantum yield or quantum efficiency, d>, which is solvent dependent, is the ratio Approximate values of quantum efficiencies are as follows naphthalene, 0.1 anthracene, 0.3 indole, 0.5 and fluorescein, 0.9. 

The appearance of a fluorescence spectrum is reminiscent of a UV-VIS spectrum. The fluorescence spectrum for a 3-substituted indole derivative is shown in Figure 6.1. The... 

Thus, knowledge of the transition moment direction of a phenol band could help in interpreting the fluorescence spectrum of a tyrosine chromophore in a protein in terms of orientation and dynamics. The absorption spectrnm of the first excited state of phenol was observed around 275 nm with a fluorescence peak aronnd 298 nm in water. The tyrosine absorption was reported at 277 nm and the finorescence near 303 nm. Fluorescent efficiency is about 0.21 for both molecules. The fluorescent shift of phenol between protic and aprotic solvents is small, compared to indole, a model for tryptophan-based protein, due to the larger gap between its first and second excited states, which resnlts in negligible coupling . ... 

A substantial literature exists describing experimental and theoretical studies of the photophysical processes of indoles. In part, this reflects the well-established use of the magnitude of the Stokes shift seen in fluorescence spectrum of the indole chromophore to probe the local environment of tryptophan residues in biological molecules [4]. However, it also reflects the complexity of indole photophysics and the fact that some aspects remain controversial. Only an overview is presented here, because a proper discussion of the photophysics of the indole ring would easily fill this chapter. Unfortunately, no recent, comprehensive review of the topic is available however, early publications have been summarized [4,5], and recent papers in this area provide leading references and excellent brief reviews of subsequent work [6]. 

Quantum yield of the tyrosine in 029 SSB is obtained by comparing the steady-state fluorescence spectrum to that of aqueous solutions of 5-methoxy-indole (5 MeOI) of known quantum yield ( Oref = 0.28) (Hersberger and Lumry, 1976). Of of 029 SSB = 0.065. This Of value is unusually high for proteins that lack tryptophan in their primary sequence such as insulin for example. Since Of of 029 SSB is approximately 70% of that of A-acetyl-L-tyrosine amide, the authors concluded that 029 SSB tyrosines do not appear to maintain strong interactions with odier residues of the protein... 

Unfortunately, no lead sensors have been developed to date that meet all of these criteria. The system that has been used most extensively to quantitate lead levels in vivo is the fluorescent sensor Indo-1 (Fig. 24) (443 45). Although originally developed as a calcium dye (446) log iCfc[Ca(II)] = 6.6, Indo-1 (2-[4-[bis(carboxymethyl)amino]-3-[2-[2-[bis(carboxymethyl)amino]-5-methyl-phenoxy]ethoxy]phenyl]-lH-indole-6-carboxylic acid) binds lead quite tightly (log ffo[Pb(II)] = 10.5 and exhibits a very different fluorescence emission spectrum when bound to lead than when free in solution or bound to calcium (Fig. 24). As a result, Indo-1 can be used to determine whether lead is present in cells, even in the presence of excess calcium, provided that the fluorescence spectrum is deconvoluted to account for calcium interference and all of the possible equilibria are taken into consideration (443, 445). The main drawback of the Indo-1 detection system is that the dye is almost completely quenched when bound to lead (Fig. 24, spectrum b), making it difficult to quantitate the amount of free lead present. 

The Indoles have also been shown to form exclplexes (solute-solvent excited-state complexes) with a wide range of electrophilic and nucleophilic compounds (32). Exclplex formation is especially evident In polar solvents. A fluorescence red shift from nonpolar to polar solvents and the concomitant loss of the vibrational structure of the fluorescent spectrum with no corresponding shift In the Indole absorption spectra accounts for this postulated exclplex formation. Hershberger and Lumry (33) suggest that some charge-transfer mechanism stabilizes the excited complex and Is a likely precursor to electron ejection. 

In order to obtain independent evidence for the involvement of the cyclodextrin cavity, fluorescence measurements were carried out for copper(II) ternary complexes with L- or D-tryptophan. In fact, the fluorescence spectrum of tryptophan has already been shown to be sensitive to the polarity of the microenvironment in which it is located and has been used in many studies as a probe for the conformation of proteins and peptides [53]. As for many fluorophores, the indole fluorescence of Trp is quenched by the copper(II) ion this effect has been used as a measure of the stability constants of copper(II) complexes [54, 55]. In a recent work, it has been shown that the fluorescence of dansyl derivatives of amino acids undergo enantioselective fluorescence quenching by chiral copper(n) complexes and that fluorescence measurements can be used for the study of enatioselectivity in the formation of ternary complexes in solution [56]. Bearing this in mind, we performed the same type of experiments by adding increasing amounts of the [Cu(CDhm)] + complex to a solution of D- or L-tryptophan [36]. The fluorescence titration curve shows that the artificial receptor inhibits the indole... 

Fig. B5.2.1. Corrected excitation spectrum (broken line) and excitation polarization spectrum of indole in propylene glycol at -58 °C. The fluorescence is observed through a cut-off filter (Corning 7-39 filter) (reproduced with permission from Valeur and Weber3 ).

Indole in cyclohexane and ethanol is excited at 270 nm populating a mixture of the La and Lb states. The La and Lb states differ in their spectral structure (Fig. la)) and Stokes shifts [3]. The unstructured spectrum of the La state shows a large Stokes shift due to the large change of the dipole moment upon electronic excitation. The dipole moment of the Lb state is similar to the ground state value. In nonpolar solvents like cyclohexane, the Lb state is energetically below the La state and its emission spectrum exhibits vibronic structure. In the polar solvent ethanol state reversal occurs after the electronic excitation and the La state becomes responsible for the more red shifted fluorescence [4],... 

A fluorescence emission spectrum is a record of fluorescence intensity vs wavelength for a constant intensity of exciting light. Excitation and emission spectra for a flavin and for the indole ring of tryptophan are both given in Fig. 23-13. The heights of the... 

Fig. 3 Flowchart for the interpretation of both spectral data and OPA-fluorescence response. Band 1 spectral band (201-207 nm) due to both the peptidic bonds and the carboxy-terminal group of the peptide molecule. Band 3 spectral band (276-280 nm) due to the respective hydroxyphenyl and indol groups of the tyrosine and tryptophan residues. cC.i. convexity interval (distance between the inflection points before and after the maximum in the original spectrum). dMiw minimum in the original spectrum. (From Ref. 104a, with permission.)...

Fusion of a benzene ring to a heterocyclic nucleus causes a bathochromic shift and increases the intensity of absorption. Thus, quinoline, isoquinoline, indole and quinolium and isoqumolium ions fluoresce in the ultra-violet. Acridine, acridone and carbazole exhibit visible fluorescence. Porphyrin18 and some of its derivatives are characterized by narrow well-defined vibrational bands and their fluorescence bands are in the red region of the spectrum. 

The complexation of indole with a- and fi-CD caused a red shift of the absorption spectrum and a blue shift of the fluorescence emission. In the case of a-CD as complexing agent, the absorption variations were described by a sequential formation of 1 1 (K =2M ) and 2 1 (Aj = 210M ) complexes. Fluorescence quenching by CsCl was wavelength dependent and lower in presence of CD than in bulk water. This was attributed to dififerent absorption spectra for 1 1 and 2 1 complexes and to dififerent levels of protection offered by the two complexes against quenching by CsCl [254]. 

Valeur, B. and Weber, G. 1977, Resolution of the fluorescence excitation spectrum of indole into the Eg and Lb excitations bands. Photochem. Photobiol. 25, 441-444. 

The complex anisotropy spectrum of indole was used to determine the absorption spectra corresponding to the 5 — L, and So—i La transitions. We present this example because of its didactic value and its importance for a detailed understanding of the fluorescence from tryptophan residues in proteins (Chapter 16). At any excitation wavelength X, the observed anisotropy is... 

The indole group of tryptophan (Trp) has a higher molar extinction coefficient than the phenolic and phenyl side chains of tyrosine (Tyr) and phenylalanine (Phe), respectively (see Table 1, Chapter 12). Thus although its quantum yield is similar to that of lyr, its fluorescence emission is much mote intense. Furthermore, its excitation spectrum overlaps the emission spectrum of Tyr and therefore fluorescence resonance energy transfer (FRET, see Chapters 2 and 3) from lyr to Trp occurs readily when both residues are in close proximity (i.e. located in the same protein molecule) and favourably orientated. The intrinsic fluorescence of a protein is therefore dominated by the contribution from Trp... 

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