N-acetyl-L-tryptophanamide Research Articles

Role of Charged Amino Acids in Sullying the Fluorescence of Tryptophan or Conjugated Dansyl Probe in Monomeric Proteins.

When Trp/dansyl probe conjugated to a monomeric protein is photoexcited, it is assumed that all emitted fluorescence originates solely from them. In this work, we show that hidden unconventional intrinsic chromophores (called ProCharTS) that originate from confined charge clusters in the protein can contaminate Trp/dansyl emission. Previous work has shown that charge recombination among charge-separated excited states of monomeric proteins, rich in charged residues, can emit weak luminescence (300-700 nm) overlapping with ProCharTS absorption (250-800 nm) and Trp (300-400 nm) and dansyl (400-600 nm) emission. We examine how this overlap taints the fluorescence arising from Trp/dansyl. We compared the effect of dense aqueous solutions of amino acids, Lys/Glu/Asp/Arg/His, on the fluorescence intensity decay/spectrum of N-acetyl-l-tryptophan amide (NATA). Significant broadening on the red side of Trp emission spectrum was observed solely in the presence of lysine, which appeared to be the most potent in altering the mono-exponential fluorescence decay of NATA. Interestingly, NATA in the presence of proteins α3C and dehydrin (DHN1), which are rich in Lys residues, showed substantial deviation from mono-exponential fluorescence decay in contrast to PEST wt and Symfoil-4P pv2, which lack Lys residues. Remarkably, Trp emission spectra among charge-rich proteins like α3W, PEST M1, and DHN1 CW1 were altered on the red side of Trp emission. Emission spectrum of dansyl-labeled human serum albumin (HuSA) was broadened and its fluorescence quenched with gradual addition of excess unlabeled HuSA, which displays bountiful ProCharTS luminescence. Our results unveil the additive influence of ProCharTS luminescence on Trp/dansyl emission with no measurable evidence of energy transfer.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Fluorescence and quantum mechanical approach on the interaction of amides and their role on the stability and coexistence of the rotamer conformations of L-tryptophan in aqueous solution.

Photophysical investigation on the fluorescence decay characteristics of L-tryptophan and a derivative N-acetyl-L-tryptophanamide (NATA) with alkyl amides were carried out in water. L-tryptophan exists in the zwitterionic form and exhibits a biexponential lifetime which is correlated to the existence of rotamer structures. Addition of formamide (F) and dimethylformamide (DMF) results in a decrease in the fluorescence lifetime and its proportion of the most stable structure of L-tryptophan wherein acetamide (ACM) results in an increase of the same. Interestingly, all the amides result in the formation of the lifetime of the rotamer whose lifetime doesn't exist initially and the lifetime and its distribution increases irrespective of the nature of amide. The interaction between L-tryptophan and amide is attributed to hydrogen-bonding such that these interactions influence the relative proportion of the existence of individual rotamers in the presence of amides.Strikingly, in the case of NATA that does not exhibit rotamer structures; the fluorescence lifetime is quenched in the presence of F, whereas ACM and DMF result in a larger fold of enhancement resulting in two different lifetimes. The variation in the fluorescence lifetime and amplitude of the various conformers of L-tryptophan and of NATA is completely governed by the concentration of the amides in solution such that the microenvironment surrounding the fluorophores are completely reorganised. The hydrogen-bonding functional groups in amides that are responsible for the coexistence of rotamers are elucidated and well supported by quantum mechanical (QM) studies. Time-correlated single-photon counting(TCSPC) technique is used as a probe as well as marker in establishing the variation in the lifetime properties of L-tryptophan and NATA with non-fluorescent hydrogen-bonding solutes in water which promotes this as fascinating field of research in the context of fluorescence properties of a complicated amino acid-like tryptophan.

Unraveling the Origin of Multi-Exponential Fluorescence Intensity Decay of Tryptophan in Proteins

The multi-exponential nature of tryptophan fluorescence intensity decay among proteins has puzzled biophysicists over all these years. Its origin has been previously attributed to, emission from multiple electronic absorption bands, namely La and Lb states; presence of a mixture of Trp rotamers that possess unique fluorescence lifetimes or multiple conformational states of the protein. However, none of the above explanations have been proved conclusive. Here, we report that the multi-exponential decay originates due to additional luminescence contribution from protein charge transfer states. We have previously shown (Chem. Sci., 2017, 8, 5416) the existence of weak but significant electronic absorption between 250—800 nm among proteins rich in charged residues resulting in Protein Charge Transfer Spectra (ProCharTS). Separated charges in the protein excited state created by photoinduced electron transfer emit weak luminescence on charge recombination. We observed that fluorescence intensity decay of N-Acetyl-L-Tryptophanamide (NATA) on excitation at 295 nm showed gradual deviation from mono-exponential decay kinetics with increasing presence of different charged amino acids (D, E, K, H and R). Notably contribution from a ∼2 ns component showed a significant increase in presence of charged amino acids at expense of ∼3 ns component arising from indole. The presence of charged amino acids also quenched its fluorescence lifetime (from ∼3 to ∼ 2.3 ns). Further, influence of ProCharTS was not restricted to intrinsic fluorophore like Trp. A charge rich protein like human serum albumin on conjugation with Dansyl (2-dimethyl amino naphthalene 6-sulphonyl chloride) probe displays a low mean fluorescence lifetime of ∼6.5 ns, while the same probe on conjugation to relatively less charge rich protein like hen lysozyme displays a lifetime of ∼14 ns. Thus, ProCharTS emission has a significant role in origin of multi‒exponential decay kinetics of Trp in proteins.

Maslinic acid conjugate with 7-amino-4-methylcoumarin as probe to monitor the temperature dependent conformational changes of human serum albumin by FRET.

Synthesis, characterization and spectroscopic investigation of maslinic acid labeled with fluorescent 7-amino-4-methylcoumarin is reported. It was found that the coumarin-maslinic derivative (MaCo) forms an excellent fluorescence resonance energy transfer (FRET) pair with the tryptophan (Trp) residue of human serum albumin (HSA). This feature allowed for monitoring HSA conformational alterations by measuring the distance between donor (Trp) and acceptor (MaCo) through Förster energy transfer mechanism. Displacement experiments confirmed that MaCo binds to subdomain IIA of HSA with independence of temperature. It was observed that, in the temperature range 35-45 °C, the fluorescence emission maximum of HSA-MaCo complex decreased, whereas in the range 45 °C-65 °C, an increment was detected. The concomitant change in the polarity of environment surrounding Trp was confirmed by red edge excitation shift experiments. Thermal denaturation of HSA was followed by time-resolved fluorescence spectroscopy. Average lifetime of Trp residue decreased with temperature due to the increment of solvent collisions and changes in the solvent exposure of Trp. To discriminate the importance of each effect, lifetime of N-Acetyl-L-tryptophanamide (NATA) at different temperatures was measured. Circular dichroism (CD) studies confirmed the loss of secondary structure of HSA with increasing temperature and showed a different trend in the conformational transformation below and above 45 °C, in agreement with steady-state and time-resolved fluorescence experiments.

Preferential interactions between protein and arginine: Effects of arginine on tertiary conformational and colloidal stability of protein solution

The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals’ forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process.

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.

Fluorescence anisotropy of tyrosinate anion using one-, two- and three-photon excitation: tyrosinate anion fluorescence.

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λmax 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at −60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos6 θ to cos2 θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.

Photophysics of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA): Heavy atom effect

Previously reported flash photolysis studies showed that the triplet state lifetime of aqueous indoles is μs long (12.5μs for tryptophan [10]), while other recently reported phosphorescence lifetimes of aqueous indoles, determined from photon counting phosphorescence techniques, vary from μs (approximately 40μs [11]) to ms (5ms for indole [12]). This study was motivated to explain the discrepancy regarding the intrinsic triplet state lifetime of aqueous indole and its derivatives: tryptophan and N-acetyl-l-tryptophanamide (NATA). For this purpose, a new methodology based on both fluorescence and phosphorescence decay kinetics incorporating the heavy atom effect have been applied in order to determine some quantitative parameters of the photophysics of indole and its derivatives. Additionally, we have also determined the triplet state lifetimes of the studied indoles using flash photolysis in which contributions from both a first order component and a second order component (from triplet–triplet annihilation) have been taken into account in the triplet state depopulation. The measured phosphorescence lifetime of the indoles examined measures between the values reported by Fischer and Strambini and is consistent with the triplet state lifetime determined from flash photolysis. We hope that the results obtained in this paper would be helpful for deriving structural and dynamical information from phosphorescence data of tryptophan residues in proteins.

Double plasmonic profile of tryptophan–silver nano-crystals—Temperature sensing and laser induced antimicrobial activity

Surface plasmon resonance (SPR) for spherical shaped silver nanoparticles showing double maxima at ∼390nm and ∼520nm respectively is reported. Self assembly of silver nanoparticles grown on tryptophan template leads to emergence of equal intensity double plasmon resonance (EIDPR). While for rod shaped nano-forms such double plasmon is explainable but for spherical shaped forms, such double plasmon can be explained on the basis of bidirectional formation of silver cluster in which attachment of silver at two nitrogen atom locations of tryptophan molecule seems to be obligatory. The absence of double resonance in case of silver nanoclusters formed with other amino acids or N-acetyl l-tryptophanamide (NATA), where bidirectional NH2 attachment is not possible, validates the proposed EIDPR mechanism. Electron micrograph of EIDPR particle indicates a bi-periodic fringe pattern indicating unusual crystalline property. Apart from sensing tryptophan, the double plasmon peaks are sensitive to temperature. Furthermore, the particle can be used as a smart killing agent showing bactericidal activity only upon exposure to low power laser.

Analysis of tryptophan oxidation by fluorescence spectroscopy: Effect of metal-catalyzed oxidation and selected phenolic compounds

Abstract The suitability of using fluorescence spectroscopy to assess tryptophan (TRYP) depletion by quantifying the loss of natural TRYP fluorescence during metal-catalyzed oxidation (MCO) was assessed. To analyze the effect of the oxidation conditions, N -acetyl- l -tryptophanamide (NATA) solutions were oxidized in the presence of Cu 2+ and Fe 2+ (40 and 80 μM), with and without 0.8 mM H 2 O 2 . Also, the effect of selected phenolic compounds (PhC), namely, catechin, genistein, quercetin, rutin, gallic acid and chlorogenic acid (0.08, 0.8 and 8.0 μM), on TRYP MCO was studied. Oxidation systems catalyzed by Fe 2+ had a major oxidative potential as a result of a higher amount of free radicals formed through the Fenton reaction. PhC could exert both antioxidant and pro-oxidant effects depending on their concentration, chemical structure of the PhC and the oxidation system. In general, all PhC acted as pro-oxidant agents at the highest dose. The major pro-oxidant effect was displayed by gallic and chlorogenic acid in the presence of Fe 2+ , Cu 2+ or Cu 2+ /H 2 O 2 . Quercetin, rutin and gallic acid showed antioxidant effect against TRYP oxidation at low concentrations in Fe 2+ /H 2 O 2 systems. Plausible mechanisms for the antioxidant and pro-oxidant effects of PhC on TRYP oxidation are discussed in the present article. The antioxidant efficiency of PhC or PhC-rich extracts must be studied before being used as functional food ingredients.

Feasibility of Using Bimetallic Plasmonic Nanostructures to Enhance the Intrinsic Emission of Biomolecules

Detection of the intrinsic fluorescence from proteins is important in bio-assays because it can potentially eliminate the labeling of external fluorophores to proteins. This is advantageous because using external fluorescent labels to tag biomolecules requires chemical modification and additional incubation and washing steps which can potentially perturb the native functionality of the biomolecules. Hence the external labeling steps add expense and complexity to bio-assays. In this paper, we investigate for the first time the feasibility of using bimetallic nanostructures made of silver (Ag) and aluminum (Al) to implement the metal enhanced fluorescence (MEF) phenomenon for enhancing the intrinsic emission of biomolecules in the ultra-violet (UV) spectral region. Fluorescence intensities and lifetimes of a tryptophan analogue N-acetyl-L-tryptophanamide (NATA) and a tyrosine analogue N-acetyl-L-tyrosinamide (NATA-tyr) were measured. Increase in fluorescence intensities of upto 10-fold and concurrent decrease in lifetimes for the amino acids were recorded in the presence of the bimetallic nanostructures when compared to quartz controls. We performed a model protein assay involving biotinylated bovine serum albumin (bt-BSA) and streptavidin on the bimetallic nanostructured substrate to investigate the distance dependent effects on the extent of MEF from the bimetallic nanostructures and found a maximum enhancement of over 15-fold for two layers of bt-BSA and streptavidin. We also used finite difference time domain (FDTD) calculations to explore how bimetallic nanostructures interact with plane waves and excited state fluorophores in the UV region and demonstrate that the bimetallic substrates are an effective platform for enhancing the intrinsic emission of proteins and other biomolecules.

Quasi-Static Self-Quenching of Trp-X and X-Trp Dipeptides in Water: Ultrafast Fluorescence Decay

Time-resolved fluorescence decay profiles of N-acetyl-l-tryptophanamide (NATA) and tryptophan (Trp) dipeptides of the form Trp-X and X-Trp, where X is another aminoacyl residue, have been investigated using an ultraviolet upconversion spectrophoto fluorometer with time resolution better than 350 fs, together with a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. We analyzed the set of fluorescence decay profiles at multiple wavelengths using the global analysis technique. Nanosecond fluorescence transients for Trp dipeptides all show multiexponential decay, while NATA exhibits a monoexponential decay near 3 ns independent of pH. In the first 100 ps, a time constant for the water "bulk relaxation" around Trp, NATA and Trp dipeptides are seen near 1-2 ps, with an associated preexponential amplitude that is positive or negative, depending on emission wavelength, as expected for a population conserving spectral shift. The initial brightness (sub-picosecond) we measure for all these dipeptides is less than that of NATA, implying even faster (<200 fs) intramolecular (quasi-) static quenching occurs within them. A new, third, ultrafast decay, bearing an exponential time constant of 20-30 ps with positive amplitude, has been found in many of these dipeptides. We believe it verifies our previous predictions of dipeptide QSSQ ("quasi-static self-quenching")-the loss of quantum yield to sub-100-ps decay process (Chen, R. F.; et al. Biochemistry 1991, 30, 5184). Most important, this term is found in proteins as well (Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214; Biophys. J. 2008, 94, 546; 2009, 96, 46a), suggesting an ultrafast quenching mechanism must be common to both.

Metal-Enhanced Intrinsic Fluorescence of Proteins on Silver Nanostructured Surfaces toward Label-Free Detection

In recent years metal-enhanced fluorescence (MEF) using silver particles has been reported for a number of fluorophores emitting at visible wavelengths. However it was generally thought that silver particles would always quench fluorescence at shorter wavelengths. We now report the observation of metal-enhanced fluorescence of the tryptophan analogue N-acetyl-L-tryptophanamide (NATA) on silver nano-structured surfaces. NATA is a model for the intrinsic tryptophan emission from proteins. We have also studied the effects of silver nanostructures on the emission of N-acetyl-L-tyrosinamide (NATA-tyr). In the case of NATA we observed increased emission, decrease in fluorescence lifetimes, and increase in photostability when NATA was embedded in 15 nm thick spin-casted poly(vinyl alcohol) film on silver nanostructured surfaces. We have also investigated the effects of silver nanostructures on the emission from thin poly(vilnyl alcohol) films containing NATA-tyr. However, we have observed no increase in fluorescence signal for NATA-tyr on silver nanostructures. To understand these results we performed numerical calculations using the Finite-Difference Time-Domain (FDTD) technique to model a tryptophan-wavelength dipole near a spherical silver particle. Our calculations reveal an enhancement of the power of the radiated emission by the excited-state fluorophore in proximity to a 100 nm silver nanoparticle covering the emission spectra of NATA and NATA-tyr. These calculations show a clear wavelength dependence with the specific spectral region displaying low-enhancement at the shorter NATA-tyr wavelength and higher enhancement at NATA emission wavelength. Our FDTD calculations also reveal that excited fluorophores in the near-field of a 100 nm silver nanoparticle can induce enhancement fields of varying degrees of the intensity of the near-fields around the particle that is dependent on the wavelength of the emission. We believe this enhanced near-fields play a role in our observation of MEF from metal surfaces. The enhanced emission of NATA on silver nanostructures suggests that the extension of MEF to the UV region opens new possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores towards label free detection of biomolecules.

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.

Peptide Sequence and Conformation Strongly Influence Tryptophan Fluorescence

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.

Surface Plasmon‐coupled Polarized Emission of N‐Acetyl‐l‐Trytophanamide¶

AbstractWe report an observation of ultraviolet (UV) surface plasmon‐coupled emission (SPCE) of N‐acetyl‐l‐tryptophanamide (NATA). The sample was spin coated from poly(vinyl alcohol) (PVA) solution on 20 nmaluminium film deposited on a quartz substracte. The directional UV SPCE occurs within a well‐defined narrow angle at 52dG from the normal to the coupling hemicylinder quartz prism. The NATA directional emission is highly p polarized as expected for surface plasmon‐coupled radiation.The 10 nm protective SiO2 layer deposited on top of the aluminum film significantly neutralized the fluorophore quenching by the metal surface. SPCE of NATA demonstratees a remarkable intrinsic dispersive property–the maximum of the emission spectrum depends on the observation angle. The efficient spectral resolution of SPCE can be used in the construction of miniaturized spectrofluorometers. The observation of SPCE of tryptophan opens a new possibility for the study of many unlabeled proteins with the technique complementary to surface plasmon resonance analysis.

Surface Plasmon–coupled Polarized Emission of N-Acetyl-l-Tryptophanamide¶

We report an observation of ultraviolet (UV) surface plasmon-coupled emission (SPCE) of N-acetyl-l-tryptophanamide (NATA). The sample was spin coated from poly(vinyl alcohol) (PVA) solution on 20 nm aluminum film deposited on a quartz substrate. The directional UV SPCE occurs within a well-defined narrow angle at 52 degrees from the normal to the coupling hemicylinder quartz prism. The NATA directional emission is highly p polarized as expected for surface plasmon-coupled radiation. The 10 nm protective SiO2 layer deposited on top of the aluminum film significantly neutralized the fluorophore quenching by the metal surface. SPCE of NATA demonstrates a remarkable intrinsic dispersive property-the maximum of the emission spectrum depends on the observation angle. The efficient spectral resolution of SPCE can be used in the construction of miniaturized spectrofluorometers. The observation of SPCE of tryptophan opens a new possibility for the study of many unlabeled proteins with the technique complementary to surface plasmon resonance analysis.

The light-induced reactions of tryptophan with halocompounds.

The light-dependent reaction between N-acetyl-L-tryptophanamide (NATA) and chloroform has been examined using fluorescence, NMR and reverse phase chromatography. The emission of NATA in the presence of CHCl3 decreases at 360 nm and increases at longer wavelengths (approximately 480 nm) upon illumination with 280 nm light. The action spectrum for the formation of the 480 nm emitting product(s) has the same shape as the excitation spectra of the indole fluorophore in NATA. The pH of the solution decreases as the reaction proceeds. The reaction rate depends on the intensity of the illumination and is of the first order with respect to both [NATA] and [CHCl3]. NMR and reverse phase chromatography results demonstrate that multiple products are formed. The reaction products give new peaks between 8.9 and 10.5 ppm in the 1H-NMR that are assigned to -CHO groups, which are added to the indole ring. Some of the products react with 2,4-dinitrophenylhydrazine and thus confirm this assignment. A scheme is proposed in which the excited indole gives off a solvated electron to initiate a series of steps that yield indole derivatives in which a -CHO group has replaced a -H in the indole ring. Similar reactions are observed when 5-hydroxytryptophan, 5-fluorotryptophan or N-methylindolacetate is used instead of tryptophan or when the chloroform is replaced with other trichlorinated compounds, such as trichloroacetic acid, trichloroethanol and trichloroethane, as well as the tribrominated compound, bromoform, and the monoiodinated compound, iodoactetate.

Phase-fluorimetry study on dielectric relaxation of human serum albumin

The dielectric relaxation (DR) of human serum albumin (HSA) was studied by the method of phase-fluorometry. The protein environment of the single tryptophan in HSA shows a relatively low-speed DR of sub-ns characteristic time. This relaxation can be measured as a decaying red-shift of the time-resolved fluorescence emission spectra. The details of calculations of time–emission matrices (TEM) and comparison to the fluorescence data of the reference solution of N-acetyl- l-tryptophanamide (NATA) are also presented.