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.     

Dark interaction of 5-methoxypsoralen and 8-methoxypsoralen with erythrocyte ghosts: a fluorescence and circular dichroism study

The dark interaction of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) with plasma membranes was studied using human erythrocyte ghosts as a model. In the presence of ghosts, modifications of the fluorescence characteristics of 5-MOP were observed, together with a quenching of the fluorescence of the tryptophan (Trp) residues of membrane proteins (up to 25%). Moreover, the appearance of an induced circular dichroism indicates that 5-MOP is located in a chiral environment. In contrast, only slight effects were observed in the case of 8-MOP. It is concluded that 5-MOP molecules are located partly within chiral protein sites of the membrane in such a way that a F?rster energy transfer can occur from the Trp residues to the psoralen molecules.     

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.     

MONOFUNCTIONAL ANGULAR FUROCOUMARINS: SEQUENCE SPECIFICITY IN DNA HOTOBINDING OF 6,4,4''-TRIMETHYLANGELICIN and OTHER ANGELICINS

The sequence specificity in the photoreaction (365 nm) of 6,4,4'-trimethylangelicin (TMA) with DNA fragments of the lac I gene of Escherichia coli was studied by using DNA sequencing methodology. In order to map the sites of TMA photoaddition, we took advantage of the (3'-5') exonuclease activity associated with T4 DNA polymerase, which is blocked by bulky adducts, such as furocoumarin photoadducts. A quantitative analysis of the sites of photoaddition is reported. TMA was demonstrated to photoreact with thymine and, to a lower extent, to cytosine. AT-rich sequences and TTT sites in a GC context are the most reactive sites towards TMA whereas TA, AT, CA, AC sites are weaker sites with similar reactivity. Cytosines in alternated CG sequences are also targets of TMA photobinding. We observed a less pronounced sequence specificity of TMA than that of other psoralen derivatives already studied (Sage and Moustacchi, 1987; Boyer et al., 1988). A comparison with other furocoumarins 4,4'-dimethylangelicin (4,4'-DMA), 4'-methylangelicin (4'-MA), angelicin, 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) is also reported. The role of flanking sequence and consequently of the local conformation at the various sites of photoaddition is discussed. A preferential orientation of the TMA molecule during the intercalation in the dark is suggested. Hot alkali treatment of TMA-modified DNA did not reveal any DNA strand breakage due to photooxidized bases.     

ESTIMATION OF AN INDEX OF HYDROPHOBICITY OF DNA INTERIOR USING 5-METHOXYPSORALEN AS A FLUORESCENT PROBE

The index of hydrophobicity of DNA interior was estimated by measuring fluorescence spectra of psoralen derivatives associated with DNA. The environment around 5-MOP associated with DNA was as hydrophobic (D_k = 34) as methanol, suggesting that the molecules reside at the space between the base-pairs in B-form DNA. This is also true for 8-MOP. Thus, planar and aromatic molecules of 5- and 8-MOP are more stable in the interior of DNA than in aqueous medium due to hydrophobic affinity.     

ACTION SPECTRA AND CHROMOPHORES FOR LETHAL PHOTOSENSITIZATION OF Candida albicans BY DNA MONOADDUCTS FORMED BY 8-METHOXYPSORALEN AND MONOFUNCTIONAL FUROCOUMARINS

The red-shift of furocoumarin action spectra, compared with their absorption spectra, has been investigated. An action spectrum for 8-methoxypsoralen (8-MOP) monoadduct formation in the yeast Candida albicans has been determined. The yeast cells were initially exposed to sublethal doses of monochromatic UVA at different wavelengths. Monoadduct formation was monitored by growth inhibition induced, after washing out any unbound 8-MOP, by re-irradiation with a constant second (non-lethal) dose of 330 nm radiation. A comparison between this action spectrum and the absorption spectrum of the dark complex of 8-MOP and DNA was made. In addition, the action spectra of monoadduct formation of five monofunctional compounds including a coumarin derivative have been determined. These action spectra were compared with their respective DNA dark complex absorption spectra. In general, the peaks of the furocoumarin DNA dark complexes show a red-shift when compared with the free furocoumarin molecule and the action spectra show peaks which correspond with the peaks of the dark complexes. Such data indicate that the DNA dark complex is the chromophore for growth inhibition in yeast rather than the free furocoumarin. The similarity of the 8-MOP monoadduct formation action spectrum and 8-MOP action spectra suggests that spectral dependence for the photobiological effects (including the red-shift) is dependent on monoadduct formation rather than, as previously suggested by several authors, crosslink formation. The action spectrum for the coumarin derivative 4-methyl N-ethylpyrrolo (3,2-g) coumarin (PCNEt) correlated well with the free molecule absorption spectrum rather than DNA dark complex indicating that the free molecule is the chromophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

STUDIES ON HUMAN LENS: I. ORIGIN AND DEVELOPMENT OF FLUORESCENT PIGMENTS

Abstract
Fluorescence spectra of normal mature human lenses have been measured and at least eight species with distinct emission characteristics identified. To determine the specific photochemical and photophysical processes responsible for the origin and development of these fluorophores, emission behavior of the products generated by successive irradiation of young human lenses (3–6 y old) as well as of L-tryptophan solution have been systematically monitored. Fluorescent products that resulted from this irradiation were comparable to many of the fluorophores detected in aged lenses, indicating that light plays a major role in the development of these pigments. In addition to photogenerated species, there are other compounds in human lenses, presumably advanced glycosylated end products, with marked fluorescence properties.
Several oxidation products of tryptophan including N -formylkynurenine or its derivatives, β-carboline or its derivatives, and anthranilic acid have been identified in the mature human lens. The development of several photoproducts also was attributed to endogenous ascorbate-mediated Maillard reaction products, which undergo photoconversion by the visible light. Although some of these chromophores could act as photosensitaizers, the sensitizing efficiency of many are low. Conversely, the near-UV filtering capability of these colored compounds conceivably could protect the vitreous and retina from development of any photochemical lesion.     

ULTRAVIOLET ACTION SPECTRUM FOR FLUOROGEN PRODUCTION IN THE OCULAR LENS

Abstract— …Previous work has demonstrated that fluorescent material (360nm excitation, 440nm emission), whose concentration normally increases with age in human lenses, can be generated artificially by exposing cultured human or animal lenses to UV radiation. In the present paper we report measurements of the rate of production of this fluorescent material in rat lenses in vitro as a function of UV irradiation wavelength. A plot of the observed rate of fluorogen production normalized to constant photon flux vs irradiation wavelength shows little action at 360 or 320nm, increases sharply at 300nm, remains relatively constant in the range 300–280nm, and then exhibits a further gradual rise from 270–250nm. The results on rat lenses are compared with results reported elsewhere for tryptophan in aqueous solution. The action spectrum for photochemical destruction of tryptophan in solution closely parallels that for fluorogen production in rat lenses. This result and other evidence suggest that photochemical destruction of tryptophan might be the initial event in UV-induced fluorogen production in the ocular lens.     

LUMINESCENCE OF L-TRYPTOPHAN IN THE DRY STATE AND IN AQUEOUS SOLUTION INDUCED BY X-RAYS AND U.V.-LIGHT AT 77°K

Abstract— The luminescence from tryptophan in the dry state and in aqueous glass. induced by u.v.-light and X-rays at 77°K, has been studied. The spectra of the luminescence from the dry material, which were identical for the two types of irradiation, were attributed to the same excited singlet level as that giving rise to the fluorescence from the aqueous sample. The strong phosphorescence exhibited by the latter. could not be detected for the dry material with either type of irradiation. This was attributed to a strong quenching of the radiative triplet level occurring upon crystallization of the substance. The yield of X-ray induced fluorescence of tryptophan in aqueous glass was found to be similar to that observed in the dry state. It was concluded that there is no extensive transfer of energy between the solvent and the solute molecules contributing to the fluorescence yield of tryptophan in aqueous glass.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.     

8-METHOXYPSORALEN-PHOTOINDUCED DNA CROSSLINKS AS DETERMINED IN YEAST BY ALKALINE STEP ELUTION UNDER DIFFERENT REIRRADIATION CONDITIONS. RELATION WITH GENETIC EFFECTS

Abstract— DNA damage induced by 8-methoxypsoralen (8-MOP) plus near UV light (UVA) was analyzed in diploid yeast using the alkaline step elution technique. The presence of 8-MOP and UVA induced DNA interstrand cross-links was revealed by the increase of DNA retained on elution filters as compared to untreated controls. The fraction of DNA retained on filters increased linearly with UVA dose. The amount of cross-links was estimated from the fraction of DNA retained on filters using a dose of -radiation leading to a number of DNA strand breaks at least equivalent to the number of 8-MOP induced photoadducts.
When 8-MOP treated cells were exposed to monochromatic light, 365 nm light induced monoadducts and cross-links whereas 405 nm light induced only monoadducts. When submitting 8-MOP plus 405 nm light treated cells to 365 nm irradiation, after removal of unbound 8-MOP by washing, a portion of 8-MOP plus 405 nm light induced monoadducts was converted into cross-links. The amount of monoadducts transformed into cross-links was dependent on the dose of 365 nm irradiation up to a maximum likely to correspond to the number of suitably positioned furan-side monoadducts that could be converted into biadducts. When 8-MOP plus 365 nm light treated cells were reirradiated with 365 nm light, following the same protocol, the maximum level of cross-linking obtainable in yeast was lower than that obtained with 8-MOP in a 405 nm plus 365 nm reirradiation protocol.
In the presence of 8-MOP single exposures to 405 nm light were found to be only slightly genotoxic. However, when followed by second exposures to 365 nm light, a dose-dependent increase in genetic effects, i.e. mutation and gene conversion, was observed in parallel to the induction of DNA crosslinks. These results stress again the prominent role of DNA cross-links in the genotoxicity of 8-MOP.     

STUDIES ON HUMAN LENS: I. ORIGIN AND DEVELOPMENT OF FLUORESCENT PIGMENTS

Fluorescence spectra of normal mature human lenses have been measured and at least eight species with distinct emission characteristics identified. To determine the specific photochemical and photophysical processes responsible for the origin and development of these fluorophores, emission behavior of the products generated by successive irradiation of young human lenses (3-6 y old) as well as of L-tryptophan solution have been systematically monitored. Fluorescent products that resulted from this irradiation were comparable to many of the fluorophores detected in aged lenses, indicating that light plays a major role in the development of these pigments. In addition to photogenerated species, there are other compounds in human lenses, presumably advanced glycosylated end products, with marked fluorescence properties. Several oxidation products of tryptophan including N-formylkynurenine or its derivatives, beta-carboline or its derivatives, and anthranilic acid have been identified in the mature human lens. The development of several photoproducts also was attributed to endogenous ascorbate-mediated Maillard reaction products, which undergo photoconversion by the visible light. Although some of these chromophores could act as photosensitizers, the sensitizing efficiency of many are low. Conversely, the near-UV filtering capability of these colored compounds conceivably could protect the vitreous and retina from development of any photochemical lesion.     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.     

MUTAGENIC AND RECOMBINOGENIC ACTION OF DNA MONOADDUCTS PHOTOINDUCED BY THE BIFUNCTIONAL FUROCOUMARIN 8-METHOXYPSORALEN IN YEAST (Saccharomyces cerevisiae)

Abstract— For the same furocoumarin 8-MOP and the same total number of photoadditions, the genetic activity of DNA monoadducts and a mixture of mono- and biadducts photoinduced by the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) is compared in the yeast Saccharomyces cerevisiae. In the presence of 8-MOP, 405 nm irradiation induces only monoadducts, whereas 365 nm irradiation induces mono- and biadducts (interstrand cross-links) in DNA. This is shown by heat denaturation-renaturation experiments on calf thymus DNA treated in vitro and by alkaline step elution analysis of DNA from treated yeast cells. For the same photobinding of tritiated 8-MOP to DNA in diploid yeast, about 20 times higher doses are needed with 405 nm than with 365 nm irradiation. Re-irradiation experiments reveal that part of the monoadducts induced by 8-MOP and 405 nm irradiation can be effectively converted into DNA interstrand cross-links by exposures to 365 nm radiation after washing-out of unbound 8-MOP molecules. 8-MOP and 405 nm irradiation induce per lethal hit cytoplasmic "petite" mutations in yeast as efficiently as the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) and 365 nm irradiation, both treatments being much more efficient than 8-MOP and 365 nm irradiation. At equal survival, treatments with 8-MOP and 405 nm radiation are clearly less efficient than treatments with 8-MOP and 365 nm radiation for the induction of forward ( CAN *) and reverse ( HIS +) mutations in haploïd yeast and for the induction of mutations ( ILV +) and genetically aberrant colonies including mitotic crossing-over in diploid yeast. The two treatments are equally efficient for the induction of mitotic gene conversion. At equal photobinding of 8-MOP, the monoadducts induced by 405 nm irradiation are found less effective than the mixture of mono-and biadducts induced by 365 nm irradiation for the induction of cell killing, mutations and mitotic recombination.     

Photooxidation of lens proteins with xanthurenic acid: a putative chromophore for cataractogenesis.

The tryptophan metabolite, xanthurenic acid (Xan), is produced through a transamination reaction in high concentrations in human lenses with age and has been isolated from aged human cataractous lenses. It has appreciable absorption between 300 and 400 nm (lambda max = 334 nm), the range absorbed by the human lens. Our recent studies have shown that unlike most tryptophan metabolites in the eye, Xan is photochemically active, producing both superoxide and singlet oxygen. To determine if Xan could act as a photosensitizer and photooxidize cytosolic lens proteins, alpha-, beta- and gamma-crystallins were irradiated (lambda > 300 nm, 12 mW/cm2) in the presence and absence of Xan. Upon irradiation and in the presence of Xan, lens proteins polymerized in the order alpha > beta > gamma as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Further analysis of the photolyzed alpha-crystallin by mass spectrometry indicated that histidine, tryptophan and methionine residues were oxidized at specific positions in a dose-dependent (irradiation time) manner. In alpha A-crystallin two forms of oxidized histidine 154 were observed, 2-imidazolone and 2-oxohistidine. Our results suggest that naturally occurring Xan is a chromophore capable of photosensitization and photooxidation of lens proteins. Furthermore, this compound could play a role in age-related cataractogenesis.     

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.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

FLUORESCENCE LIFETIMES OF OCULAR LENS CHROMOPHORES

Abstract— Fluorescence lifetimes are reported for intact human lenses in vitro. Two spectral regions were investigated: The first was excited at 296nm and detected at 332 or 370nm and corresponds to emission from tryptophan residues in the lens proteins. The second spectral region was excited at 359 nm and detected at 435 nm and corresponds to non-tryptophan 'fluorogen' fluorescence. The latter displayed a constant lifetime, 3.8 ns, independent of the anatomical part of the lens excited. This value was compared with measured lifetimes for some model fluorogens. The tryptophan fluorescence lifetime (332 nm detection) was found to vary from 1.8 to 2.8 ns depending on the anatomical part of the lens excited.