PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

PHOTOLYSIS OF CYSTINE IN THE PRESENCE OF AROMATIC AMINO ACIDS

Se dan evidencias para explicar los distintos rendimientos cuánticos de fotólisis con lux u.v. para distintos residuos de cisteina en lisozima e insulina. En sistemas modelos de mezclas de aminoácidos, el aminoácido triptofano muestra un marcado aumento en el rendimiento cuántico en la fotólisis de la cisteina. La fotosensibilidad de los residuos de cisteina en la lisozima se interpretan usando la conformación terciaria de la enzima la cual provee una relación espacial entre los residuos de triptofano y cisteina.     

THE ANAEROBIC PHOTOLYSIS OF TRYPTOPHAN CONTAINING PEPTIDES

The anaerobic photolysis of either Glycine- l -Tryptophan or Glycine- l -Tryptophan-Glycine, leads to one major product. These analogous compounds are shown to be the result of deamination followed by cyclization to the 4-position of indole. Structural proof is given, and the possible mechanisms involved in the reaction are discussed.     

LASER FLASH PHOTOLYSIS OF INDOLE IN THE PRESENCE OF AMINO ACIDS

The laser flash photolysis of indole at 265 nm in the presence of glycine, proline and hydroxy proline was studied. The relative yields of c _aq, triplet state, and indole cation radical were determined in the absence and in the presence of the amino acids. The yields were determined as a function of laser intensity and the values at very low intensity were compared with the fluorescence quenching results. It was concluded that in these conditions the photoionization of indole occurs via the fluorescent state. From the curves of triplet yield vs laser intensity, the triplet quantum yield extrapolated at low laser intensity was obtained, φr = 0.55 φ 0.05, relative to the literature value of 0.15 for φ_eag. This gives φ_F+φ_eaq= 1.0 ± 0.1 at room temperature. When proline and hydroxy proline were used as singlet quenchers, the yield of In was greater than the yield of c_aq. This was considered as evidence that a fraction of the quenching processes leads to complete electron transfer from indole to the amino acids.     

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 PHOTOLYSIS OF TRYPTOPHAN WITH 337.1 nm LASER RADIATION

Abstract— Aqueous solutions of L-tryptophan were photolyzed by exposure to 337.1 nm radiation from a pulsed nitrogen laser. These data were compared with results for the 290 nm conventional-source photolysis of tryptophan. The progress of photolysis was monitored by fluorescence analysis of tryptophan. UV absorption spectroscopy, HPLC, TLC, and proton NMR spectroscopy. The loss of Trp was observed to be first order for 290 nm photolysis but of mixed order for 337.1 nm photolysis. Five photolysis products were detected by TLC analysis, including: N-formylkynurenine. kynurenine, tryptamine (detected after 290 nm photolysis but not 337.1 nm photolysis) and two unknown products. The tryptophan-containing peptides N-acetyl-tryptophanamide (NATA) and tryptophylglycine (Trp-Gly) were also observed to photolyze upon 337.1 nm laser radiation demonstrating that this phenomenon is not restricted to free tryptophan monomer.
Since Trp is not ordinarily thought to absorb U V radiation at wavelengths as long as 337.1 nm. a number of experiments were performed in an effort to determine the mechanism of photolysis at this wavelength. Evidence is presented which indicates that the 337.1 nm laser photolysis of Trp does not result from two photon absorption, dielectric breakdown, or other laser-specific processes. Instead. it is concluded that this photolysis results either from a very weak absorption tail extending to 337.1 nm in tryptophan itself or from a reaction involving an impurity sensitizer which absorbs the 337.1 nm radiation. The sensitizing impurity. if present, could not. however, be removed by preparative HPLC and could not be detected by TLC or fluorescence analysis.     

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.     

THE ANAEROBIC PHOTOLYSIS OF TRYPTOPHAN CONTAINING PEPTIDES—II

Abstract A number of trp–containing peptides (gly- l -trp, l -ala- l -trp, l -trp-gly and N-acetyl-trp) have been photolyzed under anaerobic conditions. Analysis of the resultant photoproduct mixtures suggests the intermediacy of an hydrated electron in the case of trp-gly and N-acetyl-trp. Amino acid trp peptides deaminate with a concomitant cyclization to the 4-position of the indole. This appears to occur via a short-lived radical indole complex. Little difference was detected between photoproduct mixtures using either vycor or a Corning CS-0-54 (cut off ca. 295 nm) as filters. Thus these reactions can occur both above and below the ionization point of trp.     

INDOLE N—H BOND FISSION DURING THE PHOTOLYSIS OF TRYPTOPHAN

Abstract— –Analysis of the effect of β-carotene and benzoquinone on the decay curves of flash-induced transients in chlorophyll a solutions in pyridine and isooctane indicates that the formation of quinone radicals proceeds via a ternary complex of solvent, chlorophyll and quinone. It is suggested that the solvent is the source of electrons for this process.     

FLASH PHOTOLYSIS OF AQUEOUS TRYPTOPHAN, ALANYL TRYPTOPHAN AND TRYPTOPHYL ALANINE

Abstract— Tryptophan has been flash photolysed in deoxygenated and air equilibrated solutions. The kinetics of the subsequent reactions of the tryptophyl radical and the hydrated electron have been elucidated. Oxygen has been found to reduce the primary photoionization yield, indicating a triplet state precursor, but no evidence has been found for a biphotonic process. Appreciable differences in the photoionization quantum yields have been found for tryptophan, alanyl tryptophan and tryptophyl alanine.     

DNA STRAND BREAKS IN MAMMALIAN CELLS EXPOSED TO LIGHT IN THE PRESENCE OF RIBOFLAVIN AND TRYPTOPHAN

Abstract— When mammalian cells were exposed to visible-fluorescent light or near-UV light in the medium containing riboflavin and L-tryptophan, single-strand breaks appeared in their DNA. This did not occur if either riboflavin or tryptophan was omitted from the medium. The same effect was observed when cells were added to the pre-irradiated medium, indicating that a stable photoproduct was responsible. The induced DNA lesions were shown to be equally repairable in both excision proficient and defective (xeroderma pigmentosum) human cell lines. The active photoproduct formed was shown to be hydrogen peroxide. The possible relationship between these results and the near-UV induced killing of mammalian cells is discussed.     

THE RATES OF PHOTOLYSIS OF THE FOUR INDIVIDUAL TRYPTOPHAN RESIDUES IN UV EXPOSED CALF γ-II CRYSTALLIN

Aqueous buffer solutions of the lens protein bovine gamma-II crystallin were irradiated at 295 nm in the presence of dithiothreitol to determine the individual photolysis susceptibilities of the four tryptophan residues. Reverse-phase high performance liquid chromatography was utilized to compare the tryptic peptide maps before and after irradiation. Sequence analysis of collected tryptic peptides showed that the four tryptophans in calf gamma-II crystallin. TRP-42, TRP-68, TRP-131, and TRP-157 appeared in four distinct tryptic peptides. Fluorescence and absorption (diode array) monitoring of the eluting peptides allowed assessment of the changes in peptide absorbance and fluorescence following irradiation. Tryptophan fluorescence losses of (40 +/- 15)%, (17 +/- 4)%, (35 +/- 5)% and (15 +/- 4)% were observed for the peptides containing TRP-42, TRP-68, TRP-131 and TRP-157, respectively. Thus the four tryptophans in calf gamma-II crystallin did not all photolyze at the same rate. The rate differences are presumably related to the microenvironments of the individual tryptophan residues, and this is discussed in terms of the known crystal structure of calf gamma-II crystallin.     

THE PHOTOPHYSICAL AND PHOTOCHEMICAL PROCESSES OF TRYPTOPHAN IN INTERACTION WITH POLYNUCLEOTIDES: LASER FLASH PHOTOLYSIS STUDY

Abstract— The influence of nucleotides or polynucleotides on the photophysics and the photochemistry of tryptophan (Trp) derivatives has been investigated in aqueous solutions using the 265 nm laser flash photolysis technique. In solutions containing mixtures of N -acetyltryptophanamide and uridine monophosphate (UMP) or mercurated dUMP, the Trp triplet and the hydrated electron (e_aq) are quenched at almost diffusion controlled rates by the nucleotides leading to uracil reduction. Lysyl-tryptophyl-α-lysine (Lys-Trp-Lys) forms stable complexes in solution with normal or mercurated poly(uridylic acid) [poly(U)]. In the Poly(rU)-Lys-Trp-Lys complex the Trp triplet state is completely quenched, whereas the Trp triplet formation quantum yield is enhanced in complexes with mercurated poly(U). In this last case, the 'heavy atom effect' is characterized by a shortening of the Trp triplet lifetime in agreement with low temperature experiments. Our results also show that photoionization of Trp does occur in the complexed state with both polymers. The e_aq lifetime is however longer with the complexed than with the free peptide.     

FLASH PHOTOLYSIS OF TRYPTOPHAN AND N-ACETYL-L-TRYPTOPHANAMIDE; THE EFFECT OF BROMIDE ON TRANSIENT YIELDS

Abstract— Electronic spectra of indoles are very sensitive to solvents. These solvent effects are particularly pronounced in the case of the fluorescence spectra of indoles. In order to elucidate the solvent band shifts during the relaxation of the excited states, particularly in polar solvents, the dipole moments of the excited singlet states have been estimated from the data of solvent-dependent Stokes shifts. In addition to indole, indole-5-carboxylic acid, indole-3-carboxylic acid, indole-2-carboxylic acid, 5-cyano- and 5-bromo-indoles have been investigated. All indoles showed a substantial increase in dipole moment upon excitation to the emitting state. These results are generally consistent with the Pariser-Parr-Pople SCF MO calculations.     

NMR ANALYSES OF TRYPTOPHAN-8-METHOXYPSORALEN PHOTOREACTION PRODUCTS FORMED IN THE PRESENCE OF OXYGEN

Proton-made spectroscopy was performed on solutions of l -tryptophan and 8-mcthoxypsoralen (8-MOP) in either D₂O or DMSO-d₆ in the presence of oxygen before and after irradiation with 360 nm monochromatic light. Irradiation results in the loss of hydrogen atoms at the 3. 4 and 4'. 5' positions of 8-MOP and at the indole C₂ position of tryptophan. Changes in the aliphatic regions of the spectra also occur with irradiation. It is postulated that generation of photoreaction products between 8-MOP and tryptophan involves the 3.4 and 4'.5' positions of 8-MOP and the imidazole moiety of tryptophan.
Reprint requests to: Dr J. Megaw, Laboratory for Ophthalmic Research, Emory University School of Medicine, Atlanta. Georgia 30322. USA.     

THE EFFECT OF pH ON THE AEROBIC and ANAEROBIC PHOTOLYSIS OF TRYPTOPHAN AND SOME TRYPTOPHAN-CONTAINING DIPEPTIDES

Abstract— Rates of photolysis, quantum yields of fluorescence, and fluorescence emission maxima for the dipeptides glycyltryptophan (Gly-Trp) and tryptophylglycine (Trp-Gly) and for free tryptophan (Trp) were determined under both degassed and aerated conditions in the pH range 4.5-10.0. The photolyses were performed at 25°C using 290 nm radiation from a 1000 W xenon lamp. Photolysis rates were determined by monitoring tryptophan fluorescence loss with time. It was found that Trp-Gly and free Trp showed similar behavior in that their fluorescence quantum yields and photolysis rates increased significantly above neutral pH. In contrast, the Gly-Trp fluorescence yield was smaller than that of Trp or Trp-Gly, showing no significant increase at high pH and the photolysis rate for Gly-Trp decreased with increasing pH. In comparing aerated to degassed samples, it was found that degassing had a far greater effect on the photolysis rates of Trp and Trp-Gly than on the photolysis rate of Gly-Trp especially at higher pH. But, degassing did not change the relative fluorescence quantum yields or fluorescence emission maxima of any of the three compounds. Possible mechanisms for photolysis under various experimental conditions were examined in light of the data.     

DNA NICKING BY ULTRAVIOLET RADIATION IS ENHANCED IN THE PRESENCE OF IRON and OF OXYGEN

Double-stranded covalently closed circular supercoiled DNA (ccc DNA) from plasmid pUK 9 was irradiated in vitro at denned wavelengths in the UV region (290, 313 and 365 nm). The nicking was monitored by electrophoresis on agarose gels, ethidium staining and densitometric quantitation of supercoiled and relaxed moieties. At the explored wavelengths, the dose required for introducing one nick per million phosphodiester bonds diminishes with increased concentration of added ferric iron, whereas the effect of cupric iron is practically negligible. Adding metal chelators or bubbling argon prior to the irradiation results in a dramatic increase in the dose required for introducing one nick per million phosphodiester bonds. Taken together, these results seem to indicate that iron and oxygen play a role as cofactors in the UV-induced nicking of ccc DNA in vitro.