Indole Triplet Research Articles

On the possibility of direct triplet state excitation of indole

We studied the luminescence properties of indole in poly (vinyl alcohol) (PVA) film. The indole molecules are effectively immobilized in this polymer film and display both fluorescence and phosphorescence emission at room temperature. We noticed that the phosphorescence of indole in PVA film can be effectively excited at a longer wavelength than its typical singlet to triplet population route involving intersystem crossing. The maximum of the phosphorescence excitation is about 410 nm which corresponds to the energy of indole's triplet state. Interestingly, the phosphorescence anisotropy excited with the longer wavelength (405 nm) is positive and reaches a value of about 0.25 in contrast to the phosphorescence anisotropy excited within the indole singlet absorption spectrum (290 nm), which is negative. Very different temperature dependences have been observed for fluorescence and phosphorescence of indole in PVA film. While fluorescence depends minimally, the phosphorescence decreases with temperature dramatically. The fluorescence lifetime was measured to be a single component 4.78 ns while the intensity weighted average phosphorescence lifetime with 290 nm and 405 nm excitations were 6.57 and 5.62 ms, respectively. We believe that the possibility of the excitation of indole phosphorescence in the blue region of visible light and its high anisotropy opens a new avenue for future protein studies.

A New Perspective in the Use of FeOx-TiO2 Photocatalytic Films: Indole Degradation in the Absence of Fe-Leaching

The study addresses the degradation of Indole under low intensity solar simulated light. The first evidence is presented for sputtered films degrading Indole at the solid-air interface. The co-sputtering of FeOx-TiO2 on polyethylene (PE) leads to an accelerated Indole degradation kinetics compared to TiO2-PE and FeOx-PE films. Indole degradation on TiO2-PE, FeOx-PE and polyethylene (PE) supported photocatalysts is shown to proceed without Fe- or Ti-release as detected by inductively coupled plasma mass-spectrometry (ICP-MS). This opens a new perspective for the use of Fe-containing films in the degradation of pollutants. The mechanism of charge generation by TiO2-FeOx-PE film under light irradiation as a function of the applied light intensity provides insight and is discussed in relation to Indole degradation. The photo-generated charges in the film give rise to highly oxidative radicals which were unambiguously identified by appropriate scavenging experiments. The most oxidative species was O-singlet presenting an estimated lifetime of ∼20μs and a mean-free path of ∼140nm. The probability of the Indole triplet deactivation was 0.01 and was much smaller compared to the probability of 0.99 for the Indole∗-triplet reaction with O2 dissolved in the aqueous solution. By attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), the systematic shift of νs(NH) and the νs(CC) vibration-rotational peaks were followed during Indole degradation presenting the evidence for the increase in the inter-bond distance and molecular fluidity. The increase in the film hydrophilicity during the irradiation was evaluated and seems to be necessary for the Indole degradation. An average size of ∼16.7nm of the TiO2-FeOx nanoparticles sputtered for 2min on the PE was determined by atomic force microscopy (AFM) and remained invariable during the degradation of Indole. By Energy Dispersive Wavelength (EDW) a random distribution of FeOx and TiO2 in the FeOx-TiO2-PE films was seen. This study suggests a FeOx-TiO2 heterojunction inducing interfacial charge transfer (IFCT) from FeOx to the lower-lying TiO2 trapping states.

Temperature study of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA) triplet-state quenching by iodide in aqueous solution

In this study, the temperature dependence of the measured phosphorescence lifetimes of aqueous indole, tryptophan and N-acetyl-l-tryptophanamide (NATA) between 6 and 55°C in the absence and in the presence of iodide, a suitable intersystem crossing enhancer, has been determined. The obtained results suggest the existence of one process for the temperature-dependent, non-radiative deactivation of triplet states of the aqueous indoles in the absence of iodide. This process may be associated with the high sensitivity of indole triplet state lifetime to the subtle changes in the local viscosity of the surrounding aqueous environment or may be attributed to diffusional quenching by solvent molecules and/or by possible impurities present in water. However, the steep decrease in the measured phosphorescence lifetimes of indole and tryptophan with temperature suggests that diffusion-mediated quenching processes are not prevailing. Upon increasing concentration of iodide (up to 0.1M), the obtained Arrhenius plots for the deactivation rate (1/τph) of the triplet states of the studied indoles were linear, which provided strong support for the hypothesis of the existence of one temperature dependent non-radiative process for the de-excitation of indoles triplet state. Our results showed that this process is attributed to the diffusion-controlled solute-quenching by iodide and, most probably, proceeds via reversibly formed exciplex. At concentration of iodide higher than 0.1M highly curved Arrhenius plots were obtained, which may indicate a change in the rate determining step with a change in temperature. This change most probably is associated with a transition from diffusion-controlled exciplex formation followed by rate-determining exciplex deactivation at high temperature.

The triplet-state lifetime of indole in aqueous and viscous environments: significance to the interpretation of room temperature phosphorescence in proteins.

The interpretation of room temperature phosphorescence studies of proteins requires an understanding of the mechanisms governing the tryptophan triplet-state lifetimes of residues fully exposed to solvent and those deeply buried in the hydrophobic core of proteins. Since solvents exposed tryptophans are expected to behave similarly to indole free in solution, it is important to have an accurate measure of the triplet state lifetime of indole in aqueous solution. Using photon counting techniques and low optical fluence (J/cm(2)), we observed the triplet-state lifetime of aqueous, deoxygenated indole and several indole derivatives to be approximately 40 micros, closely matching the previous reports by Bent and Hayon based on flash photolysis (12 micros; Bent, D. V.; Hayon, E. J. Am. Chem. Soc. 1975, 97, 2612-2619) but much shorter than the 1.2 ms lifetime observed more recently (Strambini, G. B.; Gonnelli, M. J. Am. Chem. Soc. 1995, 117, 7646-7651). However, we have now been able to reproduce the long lifetime reported by the latter workers for aqueous indole solutions and show that it likely arises from geminate recombination of the indole radical cation and solvated electron, a conclusion based on studies of the indole radical cation in water (Bent and Hayon, 1975). The evidence for this comes from a fast rise in the phosphorescence emission and measurements of a corresponding enhanced quantum yield in unbuffered solutions. This species can be readily quenched, and the corresponding fast rise disappears, leaving a monoexponential 40 micros decay, which we argue is the true indole triplet lifetime. The work is put in the context of room temperature phosphorescence studies of proteins.

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.

Determination of the dioxygen quenching constant for protein and model indole triplets

The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily determined at room temperature in solution by monitoring the triplet absorption or emission following an exciting laser pulse. The dioxygen triplet quenching constants, can be measured for all these molecules and compared to the analogous singlet values determined by fluorescence methods. The dioxygen triplet quenching constant ( t k q) ranged from a high of 5.1·10 9 M −1·s −1 for the exposed indole of corticotropin to a low of 0.1·10 9 M −1·s −1 for the buried indole of asparaginase. The ratio of these values with their respective dioxygen singlet quenching constants ( s k q), t k q/ s k q, ranged from 0.3 to 0.6 for aqueous exposed polypeptide indoles. For globular proteins the t k q/ s k q value is observed to be 0.2 ± 0.1. This lower value for protein indoles is not attributable to ‘bulk’ environmental or hydrogen bonding effects, since the magnitude of t k q/ s k q( = 0.5 ± 0.1) for model indoles was independent of solvent dielectric constant, polarity, and proticity. Temperature-dependence studies were done to test whether t k q could be used to characterize the nature of the protein matrix. The activation energy ( E a for t k q was found to be 11 ± 2kcal/mol for most proteins. This E a was independent of whether the indole side-chain was solvent exposed or buried in the non-aqueous protein interior. Large E a values were also obtained for model indoles, naphtalene and nalidixic acid, dissolved in water, whereas the same compounds dissolved in 95% ethanol exhibited much smaller E a values. These data, in combination with the observation that the t k q of model indoles is insensitive to changes in solvent viscosity, indicate thet dioxygen quenching at the triplet level can not be easily used to characterize the dynamics of proteins.

Determination of the acrylamide quenching constant for protein and model indole triplets.

Abstract— The decay of the indole triplet of single tryptophan‐containing proteins and model compounds can be readily measured at room temperature in aqueous solution by monitoring the triplet‐triplet absorption or phosphorescence emission following a 265 nm exciting laser pulse. The quenching action of acrylamide on the triplet excited state of indole side chains was studied in an analogous fashion to that previously done at the singlet level (Eftink and Ghiron, 1977). The acrylamide triplet quenching constant (tkq) ranged from a high of 7.8 times 108M‐1 s‐1 for the exterior indole of corticotropin (ACTH) to a low of 2 times 105 Af‐1 s‐1 for the interior indole of ribonuclease T, (RNase T,). The ratio (7) of these values with their respective acrylamide singlet quenching constants (tkq),(γ=tkq8Kq) ranged from a high of 0.22 for ACTH to a low of 0.001 for RNase T1,. Acrylamide is also an inefficient quencher of model indoles in various solvents (i.e. it has a γ less than 1). The magnitude of γ varied from a high of 0.3 in H20 to a low of 0.02 in acetonitrile, but did not correlate with viscosity, dielectric constant or polarity. The lower efficiency observed for internal indole groups can not be explained by that class of models which predict the presence of static quenching at the triplet level, since none was observed. The present results confirm the observation of Calhoun et al. of a large discrepancy between acrylamide's singlet and triplet quenching constants for buried indole side chains, but suggest that it may be largely explained by the fact that acrylamide is an inefficient quencher of the indole triplet state (1983). The magnitude of this inefficiency is probably determined by specific microenvironmental factors. Thus, unlike 8Kq, the environmentally sensitive lkH cannot be easily used to characterize the dynamics of proteins.

Characterization of the indole triplet excited state in proteins utilizing laser flash photolysis.

The triplet-triplet absorption spectrum of the sole indole side chain of human serum albumin and its decay kinetics were previously characterized, at room temperature, by using a conventional flash photolysis method [(1978) Proc. Natl. Acad. Sci. USA 75, 1172-1175]. Exploitation of this potentially useful long lived reporter group in protein studies was limited by the excessively large sample size required by that apparatus. The 265 nm laser flash instrument used in the present work avoids this problem at the price of a loss in photo-selectivity. We report that the latter concern can be mitigated. Melittin was studied first because this polypeptide contains a single aromatic residue (W-19), and because its monomeric and tetrameric forms are good models for solvent exposed and buried indole side chains of proteins. For both forms, the indole triplet and neutral radical absorption spectra could be readily time resolved and identified on the basis of shape and differential dioxygen sensitivity. The single tryptophan containing protein human serum albumin was studied next because it contains a large number of other 265 nm absorbing moieties whose transient spectra might complicate the detection of the indole triplet. These transients were shown to not interfere significantly in the wavelength region 450 nm to 600 nm, and, in contrast to the indole triplet, they were relatively dioxygen insensitive. Thus, a facile means is available by which the indole triplet of proteins may be characterized. Subsequently the question of whether this species could be detected in the presence of nuclei acid components was investigated by flashing the phage fd. The putative nucleic acid transients were shown not to interfere and the absorbance of the indole triplet was readily time resolved. The spectral assignment was persuasively confirmed by showing that the indole triplet absorption and phosphorescence emission spectra decay with the same lifetime. The present work thus provides additional evidence for the general applicability of the indole triplet excited state as a long lived intrinsic protein reporter group.

TRIPLET‐TRIPLET ENERGY TRANSFER IN P‐TRYPSIN 1 THE ROLE OF THE INDOLE TRIPLET IN THE ULTRAVIOLET‐INDUCED PHOTOLYSIS OF β‐TRYPSIN

Abstract—Detection of triplet‐triplet energy transfer in an aqueous solution of P‐trypsin is reported. This conclusion is based on the observation that a light excited phenolate side chain can sensitize the destruction of an adjacent indole side chain. The role that the indole triplet might play in the UV‐induced photolysis of /l‐trypsin is also investi‐gated. The results suggest that the UV (309 nm)‐induced inactivation of P‐trypsin is not caused by indole ring destruction but by the disruption of disulfide bonds without thiol formation.

Flash photolysis of human serum albumin: characterization of the indole triplet absorption spectrum and decay at ambient temperature.

The method of flash photolysis was used to identify the transient absorption spectrum and to characterize the decay kinetics of the indole triplet of human serum albumin. This protein was studied because it contains a single indole side chain which is deeply buried in an expandable oily region and because the phosphorescence of the homologous indole in bovine serum albumin could not be detected at ambient temperatures. The transient was identified on the following basis: (i) its triplet-triplet absorption spectrum was similar to those previously reported for indole and tryptophan; (ii) it was quenched by small quantities of oxygen; and (iii) it was photobleached by 370- to 700-nm light. In a nitrogen-saturated solution at room temperature, the indole triplet decays exponentially for more than a factor of 10 with a lifetime of 0.5 msec. These observations suggest that, because of its exponential decay and relatively long lifetime, the triplet will be more valuable than the indole singlet as an intrinsic reporter group for the study of the structure and dynamics of proteins in solution.

Laser photolysis of indole in cyclohexane

Indole in cyclohexane was studied by nanosecond laser photolysis. The fourth harmonic (265 nm, 30 ns FWHM) of a Q-switched Nd 3+-doped glass laser was used for excitation; transient absorption changes were measured by kinetic spectrophotometry. Transient absorption spectra appearing after excitation were attributed to the indole triplet state and to the indole radical, respectively. Extinction coefficients of the indole triplet were determined using triplet naphthalene as reference. From the decay kinetics, a triplet lifetime of 16 μs was obtained at low triplet concentrations. TT annihilation was observed at higher triplet concentrations, with a rate constant of 7 X 10 9 s −1 M −1. It was shown that the radical is formed in a one-photon process from the excited singlet state. A complementary study of 1-methylindole indicated that the radical is created by dissociation at the NH bond. Extinction coefficients of the indole radical were also determined. A study of indole in methylcyclohexane as a function of temperature indicated that radiationless transition from the excited singlet state to the ground state is not significant.

Flash photolysis studies of proteins and indole derivatives in solution.

Abstract— Transients obtained upon flash photolysis of a number of proteins in aqueous solution appear to derive from electron ejection from tryptophyl residue side chains. These decay by a second order process. Oxygen is an effective quencher for the protein transients but is less so for the flash‐induced signals obtained from simple indole derivatives. Experiments using other quenchers indicate that the signals are not due to an indole triplet state, but that the triplet state may be a precursor of the flash‐induced metastabie species. Compounds which bind to the active site of chymotrypsin were found to exert only non‐specific effects on the flash‐induced signals.