Photosensitized oxidation and inactivation of pyocyanin, a virulence factor of Pseudomonas aeruginosa

Pyocyanin (PyO-) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0-7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO(-)-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO- was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by approximately 50%) than the intact PyO-, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO- analog in which the -O- moiety was replaced by the methoxy group (-OMe), was resistant to oxidation, suggesting that oxidation of PyO- involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO- and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.     

Fourth Order Derivative Spectrophotometric Determination of Lead with 1,10-Phenanthroline and Rose Bengal

Abstract

Higher order derivative techniques are mainly used in deconvoluting the overlapping absorption spectra of various analytes in their determination. The procedure utilizing higher order derivatives in molecular absorption spectrophotometry essentially are based on the formation of either binary or ternary complex formation. This paper deals with fourth order derivative spectrophotometric procedure for the determination of traces of lead based on the liquid-liquid extraction of ternary ion association complex lead with 1,10-phenanthroline and rose bengal into chloroform. The ternary ion associate is stable for over 24 hours. The developed procedure is simple, rapid, reliable and allows the determination of as low as 20 ppb of lead in sea water samples.     

PHOTOCHEMISTRY OF TYPE I ACID-SOLUBLE CALF SKIN COLLAGEN: DEPENDENCE ON EXCITATION WAVELENGTH

Although previous studies have demonstrated that the predominant photochemistry of type I collagen under 254 nm irradiation may be attributed either to direct absorption by tyrosine/phenylalanine or to peptide bonds, direct collagen photochemistry via solar UV wavelengths is much more likely to involve several age- and tissue-related photolabile collagen fluorophores that absorb in the latter region. In this study, we compare and contrast results obtained from irradiation of a commercial preparation of acid-soluble calf skin type I collagen in solution with UVC (primarily 254 nm), UVA (335–400nm) and broad-band solar-simulating radiation (SSR; 290^1–00nm). Excitation spectroscopy and analysis of photochemically induced disappearance of fluorescence (fluorescence fading) indicates that this preparation has at least four photolabile fluorescent chromophores. In addition to tyrosine and L-3,4-dihydroxyphenylalanine, our sample contains two other fluorophores. Chromophore I, with emission maximum at 360 nm, appears to be derived from interacting aromatic moieties in close mutual proximity. Chromophore II, with broad emission at430–435 nm, may be composed of one or more age-related molecules. Collagen fluorescence fading kinetics are sensitive to excitation wavelength and to conformation. Under UVC, chromophore I fluorescence disappears with second-order kinetics, indicating a reaction between two proximal like molecules. Adherence to second-order kinetics is abrogated by prior denaturation of the collagen sample. A new broad, weak fluorescence band at400–420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths. Amino acid analysis indicates significant destruction of aromatic amino acids under UVC, but not under UVA or SSR. When properly understood, collagen fluorescence fading phenomena may act as a sensitive molecular probe of structure, conformation and reactivity.     

Liquid-Liquid Extraction and Higher Order Derivative Spectrophotometric Determination of Cobalt with 1,10-Phenanthroline and Rose BENGAL

Abstract

Higher order derivative techniques are mainly used in deconvoluting the overlapping absorption spectra of various analytes in their determination. The procedure utilizing higher order derivatives in molecular absorption spectrophotometry essentially are based on the formation of binary complex viz. metal reacting with a chromogenic reagent. This paper reports a fourth order derivative spectrophotometric procedure for the determination of traces of cobalt based on the liquid-liquid extraction of ternary ion - asociation complex -cobalt, 1, 10-phenanthroline, rose bengal into chloroform. The developed procedure is simple, rapid, reliable and allows the determination of as low as 6 ppb of cobalt in high purity rare earth oxides and salts.     

Photodynamic cross-linking of proteins: IV. Nature of the His–His bond(s) formed in the rose bengal-photosensitized cross-linking of N-benzoyl-L-histidine

The photodynamic (photosensitized) cross-linking of N-benzoyl-L-histidine (Bz-His) as a model system was examined as part of a continuing study of the role of His–His intermolecular cross-links in the photosensitized cross-linking of proteins. The illumination of Bz-His in the presence of rose bengal (RB) bound to water insoluble plastic beads in 0.1 M sodium phosphate buffer of pH 7.4 resulted in the covalent cross-linking of the His derivative. The main dimeric cross-linked product (1) was isolated using a preparative silica gel 60 column and purified by preparative reverse phase HPLC. The chemical structure of the cross-link was determined using MS, 2D NMR spectral methods and other standard techniques. Product 1 was found to be a dimer of two His residues between the δ2-carbon of one residue (photo-oxidized to the carbonyl functionality at the ε1-carbon) and the ε2-nitrogen of the other residue. The formation of His–His cross-links was mediated by singlet oxygen, as would be expected with RB as the sensitizer. A mechanism for the formation of the cross-link was proposed in which the first step was the 1,4-cycloaddition of singlet oxygen to the Bz-His imidazole ring to give an unstable endoperoxide. This then underwent changes followed by nucleophilic addition and the elimination of one molecule of water to give 1.     

THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN p-BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY

Abstract— p -Benzoylphenylacetyl chymotrypsin, an acyl enzyme derivative containing the benzophenone group in the hydrophobic binding pocket, was prepared and is indefinitely stable at low pH. Photolysis of this covalent derivative leads to loss of enzymic activity and incorporation of the labeling group via formation of a second covalent bond. The efficiency of the photochemical processes is exceptionally high, producing 100% incorporation and at least 92% inactivation. Analysis of active site titration data for the photolyzed enzyme show that at least two different photochemical processes must be involved. Elimination of phosphorescence emission and reduction of UV absorption upon photolysis are consistent with initial hydrogen abstraction by benzophenone triplet state, followed by radical coupling, much as has been observed for the photoreaction of benzophenone with model systems. Photoaffinity labeling of chymotrypsin is also efficiently accomplished using two benzophenone derivatives which bind noncovalently to the enzyme's active site, although the rates of labeling are somewhat less than in the covalent complex.     

Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution

An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and (*)OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e(aq)(-) no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of (*)OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm).     

Hydroxyproline Measurement by High Performance Liquid Chromatography: An Improved Method of Derivatization

Abstract

Hydroxyproline is a postsynthetic derivative of proline which is commonly used to estimate the collagen content of tissues. Values (weight %) of hydroxyproline range from ten to twenty percent in most collagen types, with Type I having 11.4 percent hydroxyproline. This fact is useful for estimating the collagen content in various acid hydrolyzed tissue samples. In this paper, the authors describe a technique which produces linear hydrolysis of collagen coupled with a sensitive ultraviolet detection scheme for the 9-Fluorenylmethoxycarbonyl Chloride(FMOC-Cl) derivative of hydroxyproline. The separation itself employs reverse phase chromatography with a sensitivity of approximately 0.12 nmoles of hydroxyproline or 0.14 μg Type I collagen.     

Photoprotection by porcine eumelanin against singlet oxygen production

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.     

PHOTOINACTIVATION OF INFLUENZA VIRUS FUSION AND INFECTIVITY BY ROSE BENGAL

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inac-ti vation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion-competent virosomes were inactivated similarly to intact virus particles. The HA_Z portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross-linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate poly-acrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or β-carotene, and was enhanced by D₂O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the pho-toactivated dye, is also discussed.     

Photoinitiating behavior of benzophenone derivatives covalently bonded tertiary amine group for UV‐curing acrylate systems

A series of benzophenone derivatives (N‐BPs) containing tertiary amine group used as hydrogen abstraction‐type (type II) photoinitiators were synthesized through the addition reaction of secondary amines with 4‐(2,3‐epoxypropyloxy) benzophenone. The chemical structures were characterized with ¹H NMR, FTIR spectroscopy, and UV spectrum measurements. The N‐BPs showed the higher absorption in 300–400 nm than benzophenone (BP). The photoinitiating activity was examined based on the photopolymerization of 1,6‐hexanediol diacrylate using photo‐DSC method. The results showed that the photoinitiating efficiency was negatively affected by the molecular structure of alkyl group connected to the tertiary amine with the order of isopropyl (N‐BPI) < methyl (N‐BPM) < ethyl (N‐BPE) < propyl (N‐BPP). Moreover, the diethanolamine‐modified benzophenone derivative (N‐BPOH) had the highest‐photoinitiating efficiency for free radical polymerization systems among the N‐BPs. Copyright © 2011 John Wiley & Sons, Ltd.     

萘基衍生物的光敏化瞬态吸收光谱

本文利用激光闪光光解技术对二苯甲酮光敏化一系列萘基烷烃衍生物的三重态—三重态吸光光谱及他们之间的三重态能量传递进行了研究. 计算了三重态能量传递速度常数和传递效率, 二苯甲酮在不同体系中的三重态寿命, 探讨了分子结构对光敏化能量传递的影响.     

一种新型的紫外吸收剂——二苯甲酰甲烷的光谱特性及光稳定能力的初步研究

本文研究了一种新型的紫外吸收剂——二苯甲酰甲烷的光谱特性。实验结果表明,在基态,它存在酮式-烯醇式的热化学平衡,且烯醇式形成六元环分子内氢键。在顺-1,4-聚丁二烯溶液光降解的比较实验中,二苯甲酰甲烷的光稳定能力和邻羟基-对正辛氧基二苯甲酮相近,并对光稳定行为的可能机理进行了研究。     

Photoprocesses of Xanthene Dyes Bound to Lysozyme or Serum Albumin

The ground and excited state processes of eosin, erythrosin and rose bengal in aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). Noncovalent protein-dye binding was analyzed by circular dichroism (CD), fluorescence and UV–Vis absorption spectroscopy. The effects of protein concentrations and pH were studied. Fluorescence quenching of the dye takes place due to binding to lysozyme and fluorescence enhancement due to low loading to BSA. The effects of proteins on the xanthene triplet state and its precursor were observed by time-resolved 530 nm photolysis. The triplet lifetime is quenched by lysozyme and prolonged by loading to BSA. Light-induced damages on both the dyes and proteins were observed under exclusion of oxygen. Photo-oxidation is efficient for lysozyme and lower for BSA. The CD signal of the eosin/BSA system is maximum at pH 4, where the photo-oxidation is minor.     

Photochemistry and photopolymerization activity of novel perester derivatives of fluorenone: a conventional and laser flash photolysis study

The photochemistry of three novel t-butylperester derivatives of fluorenone was examined and compared with unsubstituted fluorenone and a mono-t-butylperester of benzophenone using both conventional microsecond and nanosecond laser flash photolysis. On conventional microsecond flash photolysis in 2-propanol, all four fluorenone compounds gave transient absorption in the region 300–400 nm due to a ketyl radical formed from the abstraction of a hydrogen atom from the solvent by the upper excited triplet n—π* state of the fluorenone chromophore. This assignment was confirmed by a pH-dependent study on the transient absorption spectra. The nitro-t-butylperester derivative of fluorenone gave additional absorption above 400 nm due to species associated with the nitro group. No evidence for benzoyloxy radical formation could be found in non-hydrogen-atom-donating solvents with microsecond flash photolysis which is associated with homolysis of the perester groups. On nanosecond laser flash photolysis of the fluorenone compounds at 355 nm excitation in acetonitrile and hexa-fluorobenzene, transient absorptions were observed in the region 320–640 nm due to the corresponding triplet states. All the t-butylperester derivatives showed residual absorbances at longer time delays which were tentatively assigned to the corresponding benzoyloxy radicals produced by homolysis of the perester groups. In contrast, the mono-t-butylperester of benzophenone, included for comparison only, showed very weak transient absorption in the region 320–640 nm compared with that of the strong triplet of benzophenone under the same excitation conditions. The triplet absorptions and lifetimes of the fluorenone compounds were correlated with their photopolymerization activities in bulk methylmethacrylate monomer. In oxygenated solutions, the triplet absorptions of fluorenone and benzophenone were effectively quenched; however, long-lived transient growths were observed for all the t-butylperester derivatives. The intensities of these novel transient absorptions appear to correlate with the total number of t-butylperester groups in the fluorenone molecule and tentative assignments are discussed.     

A photoactivated diazopyruvoyl cross-linking agent for bonding tissue containing type-I collagen

On the basis of the earlier examples of diazopyruvoyl (DAP) groups reported by Lawton for covalent binding and cross-linking of proteins and oligopeptides and our recent demonstration that a coumaryl diazopyruvamide was used to label Type-I collagen, we have extended our investigations to the synthesis and cross-linking capabilities of a bis-DAP polyethylene glycol to cross-link Type-I collagen. The new photoactivated cross-linking agent, N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis(ethylamine) (DPD, 2), has been designed and synthesized specifically to "weld" collagenous tissues by cross-linking Type-I collagen. A working model for the photochemical welding studies of collagenous tissues was developed using gelatin strips (gel strips) composed of denatured Type-I collagen. Gel strips are transparent to near-UV and visible light, uniform in thickness, and have reproducible composition. Furthermore, the availability of nucleophilic amine sites in gel strips was demonstrated by reaction with o-phthalaldehyde, producing a fluorescent derivative of the protein. Gel strips were coated with a solution of DPD in chloroform 7 irradiated at 320-390 nm, and the resulting bonded gel strips were tested for the strength of the weld. The welds were generally brittle and had average tensile strengths that exceeded 100 N/cm2. Welds were not formed in the absence of light or DPD. Scanning electron microscopy studies revealed a pockmarked surface from severed welds. Welds of rabbit Achilles tendon were also obtained using the tethered diazopyruvamide. These welds were much weaker, having an average tensile strength of 11.95 N/cm2 for DPD-2,2'-ethylenedioxy(bis)ethylamine comonomers in the cross-linking reaction. In both studies the welds obtained by this method were significantly stronger than the controls.     

Enzyme-assisted Cell Photosensitization: A Proposal for an Efficient Approach to Tumor Therapy and Diagnosis. The Rose Bengal Fluorogenic Substrate

Abstract— Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma-derived cultured cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fl0075 orogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.     

ALTERATIONS IN ERYTHROCYTE BAND 3 ORGANIZATION INDUCED BY THE PHOTOSENSITIZER, HEMATOPORPHYRIN DERIVATIVE

Abstract— Photosensitization of erythrocytes in the presence of hematoporphyrin derivative causes cross-linking of membrane proteins. This cross-linking is associated with partial lysis of the cells and an increased susceptibility to heat-induced membrane fragmentation. The effect of photosensitization on the organization of erythrocyte band 3 was monitored using the technique of time-resolved phosphorescence anisotropy. Band 3 rotational diffusion was somewhat restricted upon photooxidation, indicating aggregation of this major integral membrane protein.     

蓝光材料2-叔丁基-9,10-二(9,9-二正丙基芴基)蒽的合成与性能表征

本文设计并合成了一种新型蒽衍生物蓝光材料2-叔丁基-9,10-二(9,9-二正丙基芴基)蒽.化合物中引入的柔性烷基链有效抑制了分子间的相互作用,使该化合物不易结晶,同时提高了化合物在有机溶剂中的溶解度.通过量子力学方法计算发现,化合物具有顺反两种稳定构型,分子的平面性差,能减弱分子间相互作用.化合物在二氯甲烷溶液中的最大荧光发射峰在443 nm,在环己烷溶液中测得荧光量子效率为0.78,固态薄膜的最大发射峰波长相对溶液有少量红移(450 nm).热失重和差热分析结果表明,该化合物具有较高的热稳定性,分解温度和玻璃化转变温度分别为365℃和126℃.     

Photochemical stability of poly(vinyl pyrrolidone) in the presence of collagen

The photochemical stability of poly(vinyl pyrrolidone) (PVP) in the presence of 1%, 3% and 5% of collagen has been studied by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetry analysis (TG) and derivative thermogravimetry (DTG). Surface properties have been studied by contact angle measurements. PVP samples and samples containing 1%, 3% and 5% of collagen were irradiated with UV light of wavelength λ = 254 nm in air for up to 24 h. The amount of gel created during UV irradiation was estimated.PVP in the presence of 1%, 3% and 5% of collagen is less stable both thermally and photochemically. Collagen enhances photochemical processes leading to crosslinking of PVP. The contact angle measurements and values of surface free energy showed that the wettability of PVP films was changed by the addition of collagen and by UV irradiation. The increase of polarity of samples indicates an efficient photooxidation on the surface upon UV irradiation.