RESUMEN
Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin.
Asunto(s)
Albúmina Sérica/química , Albúmina Sérica/efectos de la radiación , Reactivos de Enlaces Cruzados , Humanos , Modelos Químicos , Oxidación-Reducción , Procesos Fotoquímicos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/efectos de la radiación , Pterinas/química , Pterinas/efectos de la radiación , Albúmina Sérica/metabolismo , Piel/metabolismo , Piel/efectos de la radiación , Envejecimiento de la Piel/efectos de la radiación , Triptófano/química , Triptófano/efectos de la radiación , Tirosina/química , Tirosina/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Pterins are normal components of cells and they have been previously identified as good photosensitizers under UV-A irradiation, inducing DNA damage and oxidation of nucleotides. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the oxidation of another class of biomolecules, amino acids, using tryptophan (Trp) as a model compound. Irradiation of Ptr in the UV-A spectral range (350 nm) in aerated aqueous solutions containing Trp led to the consumption of the latter, whereas the Ptr concentration remained unchanged. Concomitantly, hydrogen peroxide (H2O2) was produced. Although Ptr is a singlet oxygen ((1)O2) sensitizer, the degradation of Trp was inhibited in O2-saturated solutions, indicating that a (1)O2-mediated process (type II oxidation) was not an important pathway leading to Trp oxidation. By combining different analytical techniques, we could establish that a type I photooxidation was the prevailing mechanism, initiated by an electron transfer from the Trp molecule to the Ptr triplet excited state, yielding the corresponding radical ions (Trp(·+)/Trp(-H)· and Ptr(·-)). The Trp reaction products that could be identified by UPLC-mass spectrometry are in agreement with this conclusion.
Asunto(s)
Oxidación-Reducción , Fármacos Fotosensibilizantes/metabolismo , Pterinas/metabolismo , Triptófano/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/efectos de la radiación , Nucleótidos/metabolismo , Fármacos Fotosensibilizantes/farmacología , Pterinas/farmacología , Oxígeno Singlete/metabolismo , Triptófano/efectos de los fármacos , Triptófano/efectos de la radiación , Rayos UltravioletaRESUMEN
The photodynamic effect on tryptophan methyl ester (trpME) and tryptophan octyl ester (trpOE), using the O(2)((1)Delta(g))-photosensitizers Rose Bengal (RB) and Perinaphthenone (PN) has been studied in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) by stationary photolysis and time-resolved methods. This work reports on the influence of both the site (O(2)((1)Delta(g))) generation and the location of the tryptophan derivatives (trpD), on the photo-oxidation process in a compartmentalized system. The apparent rate constant values for chemical quenching of O(2)((1)Delta(g)) by trpOE (k(r,app)), was higher in vesicles than in water. Also, the ratio between apparent reactive and overall rate constant values for the deactivation of O(2)((1)Delta(g)) (k(r,app)/k(t,app)), increases in vesicles as compared with water, when the oxidative species is generated in the lipidic region or at the interface. Nevertheless, this quotient is lower than the corresponding value in water when O(2)((1)Delta(g)) is generated in the aqueous pseudophase. For trpME, the k(r,app)/k(t,app)values in vesicles and in water are quite similar, confirming the fact that trpME is located in the water pseudophase. Results are discussed in terms of relative protection against O(2)((1)Delta(g)) attack in a microheterogeneous medium as compared with water.
Asunto(s)
Membrana Dobles de Lípidos/química , Fosfatidilcolinas , Fotólisis , Triptófano/análogos & derivados , Cinética , Fármacos Fotosensibilizantes , Triptófano/efectos de la radiación , AguaRESUMEN
A comparative study of the photosensitizing activity of advanced glycation endproducts (AGEs) prepared by incubating glucose (Glc), threose (Threo) and ascorbate (AH-) in the presence of lysine (Lys) was performed. Photochemical activity was evaluated under low oxygen pressure with the aim to simulate the conditions of the eye lens. AGE-sensitized tryptophan and AH- photodecomposition and glucose 6-phosphate dehydrogenase inactivation were studied. In all systems, glucose-derived AGEs showed the highest photosensitizing efficiency, followed by ascorbate and threose. The presence of different sensitizers in glycation products mixtures was investigated. For this purpose, Trp decomposition quantum yields were determined at 344 and 367 nm. The values obtained at 344 nm are between three and six times higher than those observed at 367 nm, confirming the presence of at least two compounds with different photosensitizing activities in the mixtures. The chemiluminescence associated with the AGE-mediated oxidation of free Trp and Trp residues in human serum albumin was also studied, and a good correlation between the emission of light and the extent of Trp decomposition was found. In conclusion, it is demonstrated that glucose derived AGEs, which can be formed in vivo in the eye lens of diabetic patients and are accumulated in elderly lenses, have a higher photosensitizing efficiency, at low oxygen pressure, than those arising from ascorbate and threose. This high efficiency is especially significant when proteins are employed as photochemical targets, indicating that protein-sensitizer interaction and the local environment around the sensitizers play an important role.
Asunto(s)
Ácido Ascórbico/efectos de la radiación , Glucosafosfato Deshidrogenasa/efectos de la radiación , Productos Finales de Glicación Avanzada/farmacología , Fármacos Fotosensibilizantes/farmacología , Albúmina Sérica/efectos de la radiación , Triptófano/efectos de la radiación , Humanos , Cristalino , Modelos Biológicos , Oxígeno , Rayos UltravioletaRESUMEN
The photodynamic effect of a cationic Zn(II) N-methylpyridyloxyphthalocyanine (ZnPc 2) and a noncharged Zn(II) pyridyloxyphthalocyanine (ZnPc 1) has been compared in both homogeneous media bearing photooxidizable substrates and in vitro using a typical Gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were analyzed in different media. Fluorescence quantum yields (varphiF) of 0.23 for ZnPc 1 and 0.22 for ZnPc 2 were calculated in N,N-dimethylformamide (DMF). The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of PhiDelta=0.56 for ZnPc 1 and 0.59 for ZnPc 2. A faster decomposition of L-tryptophan (Trp), which was used as biological substrate model, was obtained using ZnPc 2 as a sensitizer with respect to ZnPc 1. In biological medium, the E. coli cultures were treated with 10 microM of sensitizer for different times at 37 degrees C in the dark. Both ZnPcs 1 and 2 are rapidly bound to E. coli cells in 5 min and the amount of cell-bound sensitizer is not appreciably changed incubating the cultures for longer times. The recovered ZnPc 2 after one washing step is approximately 3 times higher than 1, reaching a value of approximately 3 nmol/10(6) cells. After irradiation with visible light, a higher photoinactivation of cells was found for ZnPc 2. Thus, a approximately 4.5 log (99.997%) decrease of cell survival was obtained after 30 min of irradiation. On the other hand, a very low photodamage was found for cells treated with ZnPc 1 (approximately 0.5 log). Also, these results were established by stopping of growth curves for E. coli. In the structure of ZnPc 2, the cationic centers are isolated from the phthalocyanine ring by an ether bridge, which also provides a higher mobility of the charges facilitating the interaction with the outer membrane of the Gram-negative bacteria. These studies show that cationic ZnPc 2 is an efficient phototherapeutic agent with potential applications in photodynamic inactivation of bacteria.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Indoles , Luz , Compuestos Organometálicos , Fármacos Fotosensibilizantes , Antracenos/efectos de la radiación , Cationes/química , Escherichia coli/crecimiento & desarrollo , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Isoindoles , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Fotoblanqueo/efectos de los fármacos , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Triptófano/efectos de la radiación , Compuestos de ZincRESUMEN
The photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) has been investigated in two systems: reverse micelles of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water-bearing photooxidizable substrates and on a Hep-2 human carcinoma cell line. The effect of variation in the light dose and wavelength range (360-800, 455-800, and 590-800 nm) was compared in both media. The aerobic singlet oxygen-mediated photooxidation of L-tryptophan (Trp) was used as a model of biological substrate in a micellar system. A considerable increase of the observed rate constants of Trp (k(Trp)(obs)) was noted, increasing the irradiated area of the TMP spectrum. In vitro, the survival curves of Hep-2 cells, treated with TMP, were markedly dependent on the light wavelength ranges used for irradiation. A linear behavior between k(Trp)(obs) and the photoinactivation rate of Hep-2 cells was found, indicating that the singlet oxygen (1O2 ) is the main species responsible for cell inactivation. These results contributed to an understanding of the photodynamic process yielded by this porphyrin in vitro and the sensitivity of Hep-2 cells to photodamage.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Fotoquimioterapia , Porfirinas/uso terapéutico , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Cinética , Luz , Neoplasias Hepáticas Experimentales/patología , Micelas , Oxidación-Reducción , Fotoquímica , Triptófano/química , Triptófano/efectos de la radiación , Células Tumorales CultivadasRESUMEN
We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named 1E6, 5H5, 5A8 all belonging to the IgG1 isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human cataractous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked immunofluorescent emission was observed on structures preferentially localized in the nuclei.