SPECTRAL CHARACTERISTICS AND MECHANISM OF CHEMILUMINESCENCE FROM TRYPTOPHAN SOLUTIONS INDUCED BY UV-IRRADIATION

The spectral distribution of the chemiluminescence, fluorescence and phosphorescence of tryptophan aqueous solutions irradiated with high and low pressure mercury lamps has been measured. The blue emission bands in the region of 380–520 nm observed both in the chemi- and photoluminescence, as well as an absorbance increase at 230 and 330 nm, indicate oxidative degradation of tryptophan leading to the formation of derivatives of N-formylkynurenine, xanthurenic and anthranilic acids. Red emission bands at 630 and 705 nm in the spectrum of the chemiluminescence, an enhancement of light intensity by D₂O and its decrease by NaN₃ and DABCO suggest a partial contribution of O₂(¹Δ_g) to the photooxidation and chemiluminescence of tryptophan. The enthalpy of the exergonic reactions, leading to the formation of luminescing products, was calculated to average -270 kJ-mol.     

PHOTOGENERATION OF SUPEROXIDE ION and HYDROGEN PEROXIDE FROM TRYPTOPHAN and ITS PHOTOOXIDATION PRODUCTS: THE ROLE OF 3a-HYDROPEROXYPYRROLIDINOINDOLE*

Abstract— Mechanism of the photogeneration of hydrogen peroxide and superoxide ion from tryptophan (Trp) and its photooxidation products was investigated. Near-ultraviolet irradiation of 3a-hydro-peroxypyrrolidinoindole, an intermediate in the photooxidation of Trp, has been shown to generate hydrogen peroxide efficiently under aerobic conditions. Irradiation of N-formylkynurenine in the presence of 3α-hydroxypyrrolidinoindole also produced hydrogen peroxide. The formation of superoxide ion in both reactions has been confirmed, whereas the reaction of Trp with chemically generated singlet oxygen did not produce any detectable amount of superoxide ion.     

STUDIES ON THE PHOTO DEGRADATION OF TRYPTOPHAN

Abstract— The photodegradation of tryptophan in aqueous solution increases steadily with decrease in wavelength over the U.V. range 370–230 mμ, but the action spectrum does not parallel the absorption spectrum over these wavelengths. Thus appreciable decomposition occurs at wavelengths greater than 300 mp, even though at the concentrations tested the indole ring does not absorb energy in this region, and a photosensitive auto-oxidation mechanism is suggested to account for this.
The quantum efficiency for the destruction tryptophan at 265 mμ is 0 01 at pH 4 , 5 and 7 , but increases to 0.02 at pH 12. For the acetyl, ester and amide derivatives it is 0.03 in neutral solution.
The yellowing of tryptophan solutions during irradiation is accompanied by the appearance of an absorption maximum at about 305 mμ in the differential absorption spectrum. The increase in optical density at this wavelength bears a direct relationship to the decrease in optical density at 280 mμ: the degree of yellowing is related to the degree of destruction of tryptophan. It is less pronounced in neutral solution than at higher or lower pH values.
The yellowing of N-substituted tryptophan derivatives at 265 mμ is appreciably less than that of tryptophan, whereas derivatives with a free amino group are indistinguishable from the unsubstituted amino acid in this respect.     

FLUOROMETRIC STUDY OF TRYPTOPHAN PHOTOLYSIS

Abstract— Measurements of fluorescence spectra and fluorescence intensity for tryptophan solutions at different pH show an effective decarboxylation and deamination of tryptophan molecules under UV irradiation. The nonexponential dose-relationship of decrease in total fluorescence of tryptophan solutions is due to the formation of the products retaining indole ring in the course of these reactions. Dose-relationships and quantum yields of indole ring photolysis, deamination and decarboxylation are determined for tryptophan at 254 nm irradiation. Indole ring destruction accounts for about 60% of the total photolysis of tryptophan. Decarboxylation of tryptophan is two times more effective than its deamination. In the absence of oxygen quantum yield of indole photolysis in tryptophan and in the products of decarboxylation and deamination is reduced by a factor of two and by approximately an order of magnitude, respectively. Tryptophan photolysis products which, when excited at 365 nm. fluoresce in the visible region are formed from an intermediate product of indole ring destruction.     

THE PHOTOREACTIONS OF 8-METHOXYPSORALEN WITH TRYPTOPHAN AND LENS PROTEINS*

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri-midine bases and 8-MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8-MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8-MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8-MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8-MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8-MOP. These studies strongly suggest that 8-MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.     

PROFLAVINE-SENSITIZED PHOTOOXIDATION OF TRYPTOPHAN AND RELATED PEPTIDES

Abstract— The photodynamic action of proflavine on the amino acids cystine, rnethionine, tyrosine, histidine, tryptophan and related peptides was tested in anhydrous formic and acetic acids. Only rnethionine and tryptophan were found to be photooxidized: the former, whether free or bound in a polypeptide chain, is quantitatively converted to methioninesulphoxide; the latter, when free, is fragmented to several compounds, which may be divided into two main classes: melanines and kynurenine derivatives. Whereas masking the carboxyl group of tryptophan has no effect on the photooxidation products, N-substituted derivatives of tryptophan are converted in high yields to the corre-sponding kynurenine compounds. The possible applications of the method to biologically active polypeptides are discussed.     

PHOTOCHEMICAL MODIFICATIONS OF lac REPRESSOR: EFFECT OF EFFECTORS BINDING ON TRYPTOPHAN PHOTOOXIDATION

Abstract— UV irradiation of lac repressor modifies the fluorescence of the protein and its binding to the inducer and to the operator. It has been previously shown that the total loss of fluorescence is due to photooxidation of, on average, one of the two tryptophyl residues of each protomer. The present work explains this observation by showing that N-formylkynurenine formed at one site is responsible for the quenching of fluorescence of the other tryptophan via an energy transfer process. Consequently, no photoreaction occurs for the second tryptophyl residue. Photodamage of the two tryptophyl residues (in position 201 and 220) of each protomer were assayed by spectrofluorometric titration in the pH range from 8.5 to 5. For repressor alone, both residues are equally photodamaged. In the presence of the inducer isopropyl-β-D-thio-galactoside, IPTGt, residue 220 is completely protected, and tryptophan 201 is slightly more exposed to photooxidation. In the presence of antiinducer, residue 220 is only partially protected. Our results are discussed in terms of conformational changes triggered by the two types of ligands.     

EFFECTS OF INDOLE AND TRYPTOPHAN ON FURAN OXIDATION BY SINGLET OXYGEN IN MICELLAR SOLUTIONS

Abstract— The 1,3-diphenylisobenzofuran oxidation by singlet oxygen was investigated in aqueous dode-cyltrimethylammonium chloride micellar solutions using pyrene as photosensitizer. It was found that indole and tryptophan markedly enhanced the furan oxidation in micellar solutions. This enhancement effect strongly depended on the ethanol content in micellar solutions; enhancement effect below about 0.3 mol fraction of ethanol and inhibition effect above this mole fraction. In the former ethanol range, a quenching of pyrene fluorescence by indole or tryptophan and a difference spectrum of furan between micellar solutions in the presence and absence of indole or tryptophan appeared strongly, but they were weakened in the latter ethanol range. The interactions between indole or tryptophan and pyrene and/or furan might be responsible for the enhancement effect observed. Discussion was made in connection with the micelle structure.     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

A COMPARISON OF FLUORESCENCE QUENCHING AND FLUORESCENCE DECAY STUDIES WITH TRYPTOPHAN

Abstract— The quenching of the fluorescence of aqueous tryptophan solutions has been studied as a function of emission wavelength using acrylamide as a collisional quencher. Our quenching studies are consistent with recent observations of the heterogeneity of tryptophan fluorescence, but they show a slight discrepancy when compared to certain analyses of the decay of tryptophan fluorescence in terms of two components.     

PHOTOSENSITIZATION OF METHYL LINOLEATE OXIDATION BY TRYPTOPHAN IN PEPTIDES

The ability of tryptophan in peptides to photosensitize the oxidation of methyl linoleate (ML) was evaluated. Purified ML was irradiated (λ > 270 nm) alone or in the presence of a tryptophan-containing peptide in ethanol solution. Oxidation was monitored by measuring the dienc hydroperoxides formed from ML by high performance liquid chromatography. N-acetylphenylalanyltryptophan (NAPT) and N-acetyltryptophan were about 2-fold more effective as photosensitizers than leucyltryptophan and tryptophylleucine. N-acetylphenylalanyltryptophan photolyzed in ethanol to form multiple photoproducts including N-formylkynurenine-type compounds. However, the NAPT photoproducts did not photosensitize the oxidation of ML and N-formylkynurenine was about 8% as effective as NAPT. Sodium azide partially quenched the photooxidation sensitized by NAPT.     

THE PHOTOLYSIS OF TRYPTOPHAN IN THE PRESENCE OF OXYGEN

Abstract— Quantum yields for the destruction of tryptophan by a single 500 J flash in aqueous solution have been determined over the pH range 1–13 in both air-equilibrated and nitrogen-saturated conditions. When these quantum yields are compared with the quantum yields for radical formation and photoejection of electrons, it is found that there is good agreement only for the nitrogen-saturated case. In air-equilibrated solutions of tryptophan, there is a large disparity between the measured degradation quantum yields and those for photoejection of electrons and radical formation. Oxygen, therefore, is playing a major role in the photochemical decomposition and it is proposed that the major reaction which occurs, under normal atmospheric conditions, is the reaction of the lowest triplet excited state of tryptophan with oxygen.
Preliminary photolysis-product distributions against pH are discussed, and indicate that a total of nine major products are formed in the presence of oxygen.     

THE EFFECTS OF PORPHYRIN STRUCTURE AND AGGREGATION STATE ON PHOTOSENSITIZED PROCESSES IN AQUEOUS AND MICELLAR MEDIA

The efficiency of several porphyrins at 10 μM and 83 μM as sensitizers of the photooxidation of 0.1 mM tryptophan and histidine via a singlet oxygen-mechanism was studied in pH 7.4-buffered aqueous solutions and in aqueous dispersions of Triton X-100 micelles. The porphyrins were either solubilized in the bulk aqueous medium or associated with the micellar phase, whereas the amino acids were always located in the aqueous phase. With those porphyrins, such as uroporphyrin I, meso-tetra (4-sulfonatophenyl)porphine, meso-tetra(4-carboxyphenyl)porphine and meso-tetra)N,N,N-trimethylanilinium)porphine, which are > 98% monomeric in both media, the efficiency of histidine photooxidation was independent of the site of O₂(¹Δ_g) generation, as shown by the closely similar values for the photooxidation rate constant and oxygen-consumption quantum yield in the presence and absence of Triton micelles; the same indications were provided by photokinetic experiments with tryptophan. Actually, laser flash photolysis studies showed that the micelle-incorporation of the above mentioned porphyrins brought about only minor changes in their photophysical properties, including the relative yield of O₂(¹Δ_g) generation. On the other hand, hematoporphyrin IX, its Zn²⁺-complex, and coproporphyrin III are largely aggregated in homogeneous aqueous solution; their incorporation into Triton micelles caused an increase of the triplet quantum yield and an enhancement of the oxygen-consumption quantum yield and photooxidation rate constant for both histidine and tryptophan. The lower photosensitizing efficiency of aggregated porphyrin species in comparison with the corresponding monomeric porphyrin was confirmed by measuring the initial rate and quantum yield of oxygen consumption upon irradiation of 1 mM histidine and tryptophan in the presence of different hematoporphyrin concentrations within the 0.3-100μM range.     

A NEW FLUORESCENT PHOTOPRODUCT OF TRYPTOPHAN EVIDENCED BY LONG WAVELENGTH EXCITATION OF FLUORESCENCE

Abstract— Besides the normal tryptophan (Trp) fluorescence in aqueous solution (emission maximum at 350 nm), a new emission, peaking around 380 nm, appears by long wavelength excitation. Its fluorescence yield (φ_s 0.24) is higher than that of tryptophan (φ_Trp= 0.13). The growth of this emission is observed under different experimental conditions, mainly under UV anaerobic irradiation. To explain this observation, the formation of a C₃-hydroxylated derivative is tentatively suggested.     

PHOTOLYSIS OF THE SINGLE TRYPTOPHAN RESIDUE OF EEL TROPONIN C

Abstract— The tryptophan (TRP) residue of eel troponin C was selectively degraded by direct UV irradiation, at 302 nm, in Ar-saturated solution. Depending on the absence or presence of calcium ions, this TRP residue is exposed to aqueous medium or buried in a hydrophobic environment. Tryptophan loss was determined by both absorption and fluorescence spectroscopy and by amino acid analysis. The photodegradation yield was significantly higher for the exposed TRP residue than for the buried ones. These results give more detail on previous observations on several other proteins and corroborate the predominant influence of the polarity on the photosensitivity of a TRP residue in polypeptidic structures.     

THE PHOTOLYSIS RATES OF SOME DI- AND TRIPEPTIDES OF TRYPTOPHAN

We have measured the relative rates of photolysis of free tryptophan (trp), the dipeptides Gly-Trp, Trp-Gly, Leu-Trp, and Trp-Leu, and the tripeptides Gly-Trp-Gly and Leu-Trp-Leu. The photolyses were performed in neutral 0.1 mM aqueous solutions at 25°C using monochromatic 290 nm Xe arc radiation. Tryptophan loss was monitored by absorption, fluorescence and phosphorescence spectroscopy. The rate of tryptophan fluorescence loss was found to be different in the di-and tripeptides than in tryptophan monomer. These rate differences depended on both the identity of the neighboring amino acid (gly or leu) and on the nature of the linkage, e.g., the rate of Gly-Trp photolysis was more than 10 times greater than the rate of Trp-Gly photolysis. Degassing was found to markedly reduce (factor of 8) the photolysis rates of Trp, Trp-Gly, and Trp-Leu, but degassing only slightly reduced (less than a factor of 2) the photolysis rates of the other di-and tri-peptides. Photochemical product structures were not determined, but absorption and fluorescence spectra were obtained and products could be inferred in some cases by comparison with data of previous workers. The products appeared to differ greatly among the various peptides studied; Trp, Trp-Gly, and Trp-Leu gave oxidation products, while Gly-Trp and Leu-Trp apparently gave ring closure products, not requiring oxygen.     

UVB-INDUCED PHOTOPEROXIDATION OF LIPIDS OF HUMAN LOW and HIGH DENSITY LIPOPROTEINS. A POSSIBLE ROLE OF TRYPTOPHAN RESIDUES

Ultraviolet radiation of the UVB region readily destroy tryptophan (Trp) residues of low (LDL) and high (HDL) density lipoproteins. The photooxidation of tryptophan residues is accompanied by the peroxidation of low and high density lipoproteins unsaturated fatty acids, as measured by the thiobarbituric acid assay. Moreover, low and high density lipoproteins are natural carriers of vitamin E and carotenoids. These two antioxidants are also rapidly bleached by UVB. The UVA radiation promotes neither tryptophan residue destruction nor lipid photoperoxidation. The redox cycling Cu2+ ions considerably increase lipid photoperoxidation. The synergistic action of photo and auto (Cu2(+)-induced) peroxidation induces marked post-irradiation modifications of apolipoproteins as illustrated by the degradation of most tryptophan residues after overnight incubation in the dark of pre-irradiated samples.     

QUENCHING OF PYRENE FLUORESCENCE BY TRYPTOPHAN IN MICELLAR SOLUTIONS

Deactivation of excited pyrene incorporated to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecyl sulfate) and neutral (Triton X-100) micelles by tryptophan has been investigated over a wide pH range. Data obtained allow an estimation of the tryptophan association to the micelles, of the tryptophan apparent p K in the micellar solutions, and of the dynamics of tryptophan incorporation to the micellar pseudophase. In particular, the data obtained at pH 7 allow an estimation of the effect of the micellar charge upon the binding capacity of the tryptophan zwitterion.     

LINEAR DICHROISM STUDY OF METALLOPORPHYRIN TRANSITION MOMENTS IN VIEW OF RADIATIONLESS INTERACTIONS WITH TRYPTOPHAN IN HEMOPROTEINS

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe³⁺-PPIX (ferric-heme) and Fe²⁺CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300–380 nm) and Soret (380450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300–380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300–380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J . 63, 648–653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 from the α-γ meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.     

PHOTOIONIZATION OF TRYPTOPHAN: AN ELECTRON SPIN RESONANCE INVESTIGATION

The photoionization of tryptophan has been studied by ESR spectroscopy using the free radical scavenger 5, 5 dimethyl-1-pyrroline-l-oxide as a spin trap for hydrated electrons. A special set-up has been devised to irradiate more than one aliquot of the bubbled aqueous solutions in the cavity without removing the ESR cell out of the cavity.
Quantum yields of electrons production have been estimated as a function of irradiation wavelength. No photoionization threshold has been observed in these experiments.