ULTRAVIOLET AND N-FORMYL-KYNURENINE-SENSITIZED PHOTOINACTIVATION OF BOVINE CARBONIC ANHYDRASE: AN INTERNAL PHOTODYNAMIC EFFECT

Abstract —Direct photoinactivation by UV light of bovine carbonic anhydrase, as well as its photosensitization by N -formyl-kynurenine, a tryptophan photooxidation product, have been investigated. In the presence of oxygen both methods lead to similar results: the enzyme loses its activity, the tryptophanyl, histidyl and, to a lesser extent, tyrosyl residues being destroyed. In nitrogen-saturated solutions, a dramatic drop is observed in the photoinacitivation yield under the direct action of ultraviolet light, whereas histidyl residues remain intact. Evidence indicates an internal photodynamic action of N -formyl-kynurenine in the protein core produced by the UV photooxidation of tryptophanyl residues. Photoinactivation of oxygenated enzyme solutions by external and internal photodynamic action correlates with histidyl residue destruction via singlet oxygen. The possible importance of the photodynamic ability of N -formyl-kynurenine in the photochemistry of proteins, DNA, and cells is discussed.     

INTERACTION OF PHOTODYNAMICALLY INDUCED CELL KILLING and DARK CYTOTOXICITY OF RHODAMINE 123

Abstract— Loss of clonogenicity of Chinese hamster ovary (CHO) cells, murine L929 fibroblasts and human bladder carcinoma T24 cells caused by photodynamic treatment (PDT) with hematoporphyrin derivative (HPD) is synergistically enhanced by subsequent incubation with rhodamine 123 in the dark. For CHO and L929 cells this synergistic interaction can be explained by an increased uptake of rhodamine 123 as the result of the photodynamic treatment. With aluminum phthalocyanine (AIPc) as photosensitizer only additive effects were observed in the three cell lines. Incubation in the dark with rhodamine 123, followed by a photodynamic treatment with HPD, resulted in an antagonistic interaction with regard to loss of colony formation. With AIPc the combination of treatments resulted in an additive effect with L929 and T24 cells, whereas with CHO cells a slight antagonistic interaction was observed. An antagonistic effect was also observed in model experiments, treating histidine photodynamically with HPD and measuring oxygen consumption. A possible explanation of these results could be an interaction or complex formation of rhodamine 123 with HPD resulting in a diminished singlet oxygen production. With AIPc this does not take place.     

Photoproducts Formed in the Photoyellowing of Collagen in the Presence of a Fluorescent Whitening Agent

Fibrous proteins discolor on exposure to the UV component of sunlight. This effect is exacerbated in the presence of fluorescent whitening agents (FWAs), which are often applied to textiles to improve product brightness. Tryptophan photoproducts have been identified as significant contributors to protein photoyellowing; however, the role of non–tryptophan-derived chromophores is less clear. In this study bovine collagen, containing no tryptophan residues, was irradiated in the presence and absence of the stilbene-derived FWA, 4,4'-bis(2-sulfostyryl)biphenyl (DSBP) and photoproducts were identified using mass spectrometry. Photoyellowing was found to be dependent on the presence of the FWA, attributed to amplified generation of reactive oxygen species (ROS), particularly hydroxyl radicals and peroxynitrite. Four key proteinaceous photomodifications contributing directly to photoyellowing were located in irradiated collagen pretreated with DSBP, namely dopa, nitrophenylalanine, nitrotyrosine and nitrohistidine. This represents the first direct characterization of the three nitrated residues in the photoyellowing of an isolated fibrous protein, and implicates the ROS, peroxynitrite, as a key contributor to protein photoyellowing. Direct oxidative modification of the FWA itself was also observed. This study demonstrates that, even in the absence of tryptophan residues, significant photomodification of protein residues leading to chromophore formation occurs in the presence of an FWA.     

Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.     

Studies on chemical modification of Tulipa gesneriana lectin

Modification of lysine, tyrosine, histidine, aspartic acid and glutamic acid residues did not affect the agglutinating activity of the Tulipa gesneriana lectin (TGL). Modification of two arginine residues per subunit in the lectin with either 2,3-butanedione or phenylglyoxal led to an almost complete loss of activity. An inactive lectin modified with 2,3-butanedione recovered a full activity on dialysis against Tris-HCl buffer. The presence of 0.1 M (alpha-1----6) linked mannotriose, a potent inhibitor of the lectin, protected all the arginine residues from modification and the lectin was fully active. Circular dichroism spectroscopy showed that no significant conformational change of TGL occurred following arginine modification. A treatment of the lectin solution with N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, chemical reagents for tryptophan modification, caused turbidity of the solution, accompanied with complete loss of activity. The fluorescence emission spectrum of the lectin showed a characteristic tryptophan emission with a maximum centered at 336 nm. Upon addition of manno-oligosaccharides a decrease of the fluorescence intensity was observed, indicating that the environment of tryptophan residues altered. These results suggest that arginine and tryptophan residues are importantly involved in the sugar binding of TGL.     

THE MODIFICATION OF RIBONUCLEASE-A BY NEARULTRAVIOLET IRRADIATION IN THE PRESENCE OF PSORALEN

Abstract— Ribonuclease A is inactivated when irradiated under oxygen by UV-A light in the presence of psoralen. The rate of inactivation is greatly reduced by sodium azide. ascorbate or nitrogen, whereas the substrate gives only very limited protection. A ribonuclease sample modified to 40% remaining activity presented a significant modification of amino acid residues known to be sensitive to oxidation and 1.4 mol of bound psoralen per mol of protein. The secondary structure of the enzyme, as assessed by circular dicroism was not changed by irradiation; neither was aggregation of the enzyme to a higher mol wt evident. Studies on the tryptic peptides fractionated by high performance liquid chromatography showed that the photomodification occurs with very low selectivity. All the five peptides containing hystidine, tyrosine and methionine residues were greatly modified, although two, those containing histidine residues 12 and 119 in the sequence, amino acids known to be involved in the catalytic activity of ribonuclease. are modified to a greater extent. The protein bound psoralen. revealed by radioactivity in the HPLC eluate, was not found associated to only one or few peptide peaks but spread on a large zone of elution.     

Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein

The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.     

FORMATION OF PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IN COLLAGEN TYPES I AND IV EXPOSED TO ULTRAVIOLET LIGHT

Abstract— Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.     

Separation of human orosomucoid major gene products using immobilized copper affinity chromatography and identification of the metal-interactive residues

Summary The major gene products of human orosomucoid, GP1 and GP2, were purified using immobilized copper affinity chromatography [Cu(II)-IMAC], with 3 mM imidazole as eluent. Both gene products bound to the Cu(II)-IMAC column in the presence of 1 M NaCl, but at different pHs. GP1 was not retained after treatment with diethylpyrocarbonate (DEPC). This modification was characterized using difference absorbance spectrophotometry and mass spectrometry. The latter provided unambiguous assignment of some of the modified residues. No correlation was observed between the modification of histidine/tyrosine and protein retention. Furthermore, removal of the carbethoxy groups of modified histidine and tyrosine by hydroxylamine treatment did not improve the retention. Therefore neither histidine nor tyrosine could be the critical residues in metal recognition. Results from mass spectrometric analysis of retained and unretained fractions of DEPC modified GP 1 indicated that the lysine residues 130/135 and 152 were modified significantly in both fractions, but to a relatively less extent in the retained one. We suggest that the retnetion of GP1 involves several residues including lysines, and that a critical number of these is necessary for retention.     

Improved Stability and Photodynamic Activity of Water‐Soluble 5,15‐Diazaporphyrins Incorporated in β‐(1,3‐1,6)‐d‐Glucan with On‐Off Switch

5,15‐Diazaporphyrins, which have a large absorption at wavelengths over 600 nm, were dissolved in water by complex formation with β‐(1,3‐1,6)‐d ‐glucans. Aqueous solutions of these complexes were relatively stable compared with their trimethyl‐β‐cyclodextrin‐complexed analogues. β‐Glucan‐complexed diazaporphyrins showed quenched fluorescence and had low singlet‐oxygen‐generation abilities owing to random self‐aggregation. However, external stimuli, such as the presence of liposomes or intracellular uptake, restored the fluorescence and singlet‐oxygen‐generation abilities of β‐glucan‐complexed diazaporphyrins. Consequently, β‐glucan‐complexed diazaporphyrins showed very high photodynamic activities toward HeLa cells.     

UV radiation effects on flavocytochrome b2 in dilute aqueous solution

A dilute aqueous solution of flavocytochrome b2 when exposed to inactivating doses of UV radiation at 280 nm underwent reversible loss in activity both under aerated and deaerated conditions. The active site as well as the substrate binding sites were found to be modified in the irradiated enzyme. Irradiation of the enzyme in the UV-C range resulted in partial unfolding of the polypeptide framework. Destruction and/or modification of both tryptophan and tyrosine residues as well as heme moieties took place. Preliminary laser flash photolysis studies suggest that the initial photo-ionization takes place with tryptophan and tyrosine residues with the formation of excited states and radicals, and then rapid transfer of electrons takes place to histidyl and cystinyl sites which might have eventually been altered in the process.     

New application for expanded porphyrins: sapphyrin and heterosapphyrins as inhibitors of Leishmania parasites

Sapphyrins and a series of related porphyrinoid macrocycles have been investigated as potential agents for the treatment of leishmaniasis. The effectiveness of the compounds was evaluated in vitro upon incubation with Leishmania tarentolae or L. panamensis amastigotes and promastigotes. Their effectiveness was also assessed against intracellular L. panamensis. The cytotoxicity of the compounds was evaluated in vitro using the U937 human promonocyte cell line. Effectiveness and cytotoxicity were assessed in the presence and absence of visible light to assess the photodynamic activity of the compounds. Sapphyrin and two related heterosapphyrins were shown to be particularly effective as inhibitors of Leishmania. A photodynamic effect was observed, which may be attributed to the formation of reactive oxygen species. Yields of singlet oxygen ((1)O(2)) produced were determined in ethanol solutions by direct measurement of (1)O(2) phosphorescence. Confocal microscopy demonstrated that sapphyrin and related macrocycles were taken up by the Leishmania cells and that their presence induces the formation of mitochondrial superoxide. Sapphyrins have been widely investigated as anticancer agents and we here show activity against the Leishmania parasites.     

Identification of nitroglycerin‐induced cysteine modifications of pro‐matrix metalloproteinase‐9

Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase‐9 (MMP‐9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro‐form of MMP‐9 (proMMP‐9), has not been investigated and was examined here using nano‐flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision‐induced dissociation (CID) and electron‐transfer dissociation (ETD), were employed for the identification of various modifications. A high‐resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP‐9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP‐9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr‐262, in close proximity to an oxidized Cys‐256 of proMMP‐9. The intramolecular interaction between these two residues toward NTG‐induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG. Copyright © 2011 John Wiley & Sons, Ltd.     

ALTERATION OF GUANINE RESIDUES DURING PROFLAVINE MEDIATED PHOTOSENSITIZATION OF DNA

Abstract— Mild photodynamic treatments of proflavine-calf thymus DNA complexes induce a unique and quantitatively important alteration of the guanine residues which can be related to the lethal lesions due to the combined action of proflavine and light on phages. The 'altered guanine' is destroyed by HClO₄ but is recovered after partial DNA depurination under the form of two photoproducts. The first product, G_ox, elutes as guanine on a Sephadex column but has a modified UV absorbance spectrum. It gives rise by further irradiation to another product, X, which elutes at pH 9.7 as a pyrimidine compound and presented a maximal UV absorbance at 246 nm. Product X is also selectively released by piperidine fixation onto the photo-damaged DNA. The guanine degradation process is markedly decreased in the presence of the singlet oxygen quencher, NaN₃. The photodynamic lesion inhibits the enzymatic degradation of the DNA but generates locally denatured regions that are sensitive to S1 endonuclease.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.     

Quinolone Antibiotic Photodynamic Production of 8-Oxo-7,8-dihydro-2'-deoxyguanosine in Cultured Liver Epithelial Cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lome-floxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50–400 uAf) or up to 3.6-fold in the presence of CFX (50–400 µM) and UVA (20 J/cm²) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm². Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

PHOTODYNAMIC INACTIVATION OF E. COLI BY ROSE BENGAL IMMOBILIZED ON POLYSTYRENE BEADS

Abstract. The photodynamic inactivation of E. coli by visible light and O₂ was found to occur in the presence of the sensitizer rose bengal, immobilized by covalent bonding to polystyrene beads. The demonstrated absence of significant amounts of dissolved rose bengal indicated that an inactivation mechanism based on penetration of sensitizer molecules into the cell's interior could not be operating. Survival curves typically exhibited induction periods followed by rapid exponential death, with 99.99% kill requiring 1–2 h depending on conditions. A mechanism involving the participation of photo-generated singlet excited oxygen O₂(¹δ) in inactivation of E. coli is proposed. The photodynamic inactivation rate increased significantly in H₂O compared with H₂O, which is evidence supporting singlet oxygen as an active intermediate, since O₂(¹δ) has a much longer lifetime in H₂O than in H₂O. H₂O did not act as a short term poison in the absence of sensitizer.     

Photochemical modifications of lac repressor--II. Tryptophan photochemistry as a probe in studying the allosteric behaviour of the protein

Irradiation of lac repressor under aerobic conditions in the near UV region (295-400 nm) decreases the Trp fluorescence of the protein. A total loss of fluorescence corresponds to the destruction of all tryptophanyl residues. Irradiation with light of wavelength between 250 and 400 nm quenches fluorescence completely when only half of the Trp residues ae destroyed. An internal photodynamic effect, in which N-formylkynurenine, a principal photoproduct of Trp, sensitizes further the destruction of the other Trp residues, accounts for our results. Experiments performed in the presence of sodium azide suggest that singlet oxygen is not involved in the destruction of Trp, but may be responsible for histidine degradation. Irradiating the repressor complexed with non-operator E. coli DNA has the same effect on Trp residues as irradiating repressor alone. On the contrary, when repressor is complexed to lac operator, both tryptophanyl residues seem to be destroyed simultaneously. This indicates that binding of specific operator DNA at the DNA site induces changes in the environment of the tryptophanyl residues (mainly tor Trp 220) which cannot further transfer in excitation energy to the photoproduct of the other Trp. A prolonged irradiation destroys the complex, leading to the same result observed for non-specific complex or for repressor alone. These results are discussed in terms of the proximity of Trp from the inducer binding site and the allosteric behaviour of the repressor.     

Photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen

The photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen (AcPso), proposed for the photochemotherapy of some skin diseases, were investigated. The photoreaction of AcPso with DNA is weaker in the presence of air than in a nitrogen atmosphere, in terms of total photobinding and DNA cross-linking; when UVA irradiation is performed in air, AcPso behaves as a monofunctional reagent. The quenching effect of oxygen is related to the high capacity of AcPso to produce singlet oxygen. Furthermore, it is demonstrated that AcPso photoadducts are better producers of singlet oxygen than free AcPso in solution. Using DNA sequencing methodology, two modes of DNA photosensitization by AcPso are shown, these lead to the formation of photoadducts mainly at T residues (and at C to a lesser extent) and to photo-oxidized G residues probably via singlet oxygen. Chemical or enzymatic cleavage were used as probes in these experiments. A rapid assay for the detection of the photodynamic effect of a photosensitizer on DNA, involving oxygen, is also described. Finally, the cytotoxicity and genotoxicity of AcPso on E. coli WP2 cells appear to be related to its ability to form photoadducts, in particular cross-links, rather than to its capacity to produce singlet oxygen.     

AMPLIFICATION OF PHOTODYNAMIC EFFECTS BY THE SYNERGISTIC ACTION OF UV-B and TRYPTOPHAN

Abstract The formation of singlet oxygen by photodynamic agents is shown to be notably amplified by the combination of UV-B radiations plus tryptophan in aqueous medium because of the formation of N-formylkynurenine. a tryptophan photoproduct which is also a good photosensitizer. The biological implication of these effects is discussed.