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Browsing by Author "SILVA, E"

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EFFECTS OF PH AND IONIC MICELLES ON THE RIBOFLAVIN-SENSITIZED PHOTOPROCESSES OF TRYPTOPHAN IN AQUEOUS-SOLUTION
(ELSEVIER SCIENCE SA LAUSANNE, 1991) SILVA, E; RUCKERT, V; LISSI, E; ABUIN, E
The effects of pH and ionic micelles on the rates of product formation following irradiation of riboflavin in the presence of tryptophan were investigated by absorption and fluorescence spectroscopy. Under anaerobic conditions, formation of riboflavin-tryptophan adducts was inhibited in acid solutions and by the addition of anionic (sodium dodecylsulphate) and cationic (cetyltrimethylammonium bromide) micelles. The oxidation of tryptophan photoinduced by riboflavin was considerably faster in basic solutions.
EXCITATION OF MESOPHYLL AND BUNDLE SHEATH CHLOROPLASTS OF ATRIPLEX-REPANDA IN THE ABSENCE OF LIGHT
(AMER SOC PHOTOBIOLOGY, 1991) SILVA, E; RODRIGUEZ, G; FALJONI, A; CILENTO, G
Mesophyll and bundle sheath chloroplasts isolated from Atriplex repanda cells promote oxygen consumption by isobutyraldehyde or phenylacetaldehyde. In all cases, a red emission and reduction of tetrazolium blue was observed. Addition of horseradish peroxidase greatly increases the reduction of the dye. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the reduction of the Hill acceptor was fully suppressed. This suppression was abolished when 2,6-dichlorophenolindophenol and ascorbate were added to the systems. These results indicate that, in mesophyll and bundle sheath chloroplasts, chlorophylls can be efficiently excited in the absence of light and an electron flow through the photosystems can be promoted.
EXPOSURE OF TRYPTOPHANYL RESIDUES IN ALPHA-LACTALBUMIN AND LYSOZYME - QUANTITATIVE-DETERMINATION BY FLUORESCENCE QUENCHING STUDIES
(1986) EDWARDS, AM; SILVA, E
The effect of iodide ion on the tryptophyl fluorescence of the homologous proteins lysozyme and .alpha.-lactalbumin in their native form, as well as in their modified structures and in fragments from these proteins was studied. By assessing the contribution to the total fluorescence of the exposed and buried Trp residues, and of the respective fluorescence quantum yields, the quantization of the number of Trp exposed to the solvent for all the species studied was possible. Both native proteins show an important increase in the number of Trp residues exposed to the solvent when treated with denaturing agents. The peptides L-II (aa 13-105) and .alpha.-I (aa 1-90) from lysozyme and .alpha.-lactalbumin, respectively, showed Trp residues with different degree of exposure, whereas the smaller fragments, L-III (aa 106-129) and .alpha.-II (aa 91-123), had all their Trp residues exposed to the solvent.
ISOLATION AND PHOTOOXIDATION OF LYSOZYME FRAGMENTS
(1981) FERRER, I; SILVA, E
Reduction of the 4 disulfide bonds and further carboxymethylation of lysozyme followed by its reaction with CNBr brings about L-I, (aa [amino acids] 1-12) and L-II-III (aa 13-129) peptides. When breaking the polypeptidic chain by CNBr action and freeing the peptides formed through S-S bonds reduction and carboxymethylation, 3 peptides are obtained corresponding to L-I (aa 1-12), L-II (aa 13-105) and L-III (aa 106-129). L-II-III, L-III and L-II peptides were separately subjected to photo-oxidation in presence of riboflavin in 0.05 M phosphate buffer, pH 7.0. The kinetic analysis of Trp photo-oxidation in L-II-III peptides shows that these residues keep, to a great extent, the degree of exposure they had in native lysozyme. L-II peptide also presents Trp residues with a different degree of exposure. Presence of Tyr photo-oxidation in L-II and L-II-III peptides (this does not take place in native lysozyme) suggests a relationship between photo-oxidation selectivity and the degree of exposure of certain amino acid residues in spatial configuration.
LIGHT-INDUCED BINDING OF RIBOFLAVIN TO LYSOZYME
(1977) SILVA, E; GAULE, J
Photodynamic inactivation of lysozyme in air saturated H2O and D2O (phosphate buffer 0.05 M, pH 7.0) in the presence of methylene blue and riboflavin was studied. When H2O was replaced by D2O a great increase in rate of photoinactivation of lysozyme was observed. Photooxidation was inhibited by singlet oxygen quenchers like NaN3, and these reactions may have occurred via a singlet oxygen mechanism. Riboflavin was strongly bound to the enzyme as a result of illumination. A higher quantum was observed when riboflavin was used, although this dye was bleached during irradiation.
OBTENTION OF A PHOTOINDUCED ADDUCT BETWEEN A VITAMIN AND AN ESSENTIAL AMINO-ACID - BINDING OF RIBOFLAVIN TO TRYPTOPHAN
(1987) SALIMHANNA, M; EDWARDS, AM; SILVA, E
Studies in animals have suggested that the products of the irradiation of tryptophan in the presence of riboflavin may play a role in the development of hepatic dysfunction during parenteral nutrition. In this paper we describe the formation of an adduct between tryptophan and riboflavin obtained as a consequence of an anaerobic irradiation of these compounds. Through the use of molecular sieves and of an ion-exchange resin it was possible to separate the photo-adduct from the dimer riboflavin and other reaction products. The various fractions were characteized on the basis of their absorption and emission spectra. Also used were measures of anisotropy of fluorescence emission in order to characterize the derived adduct.
PHOTOCHEMICAL REACTIVITY OF THE HOMOLOGOUS PROTEINS ALPHA-LACTALBUMIN AND LYSOZYME
(1985) EDWARDS, AM; SILVA, E
The fluorescent behavior and the photodynamic effect was studied in native and structurally modified lysozyme and .alpha.-lactalbumin. The Tyr residues in lysozyme and .alpha.-lactalbumin show different sensitivities to the photodynamic effect. The effect is zero in the case of Tyr from native lysozyme. In contrast, the Tyr residues in .alpha.-lactalbumin are susceptible to photooxidation, which indicates a greater degree of exposure to the solvent. The 3 His residues of .alpha.-lactalbumin have different degrees of exposure and show 2 different kinetics of photooxidation whereas the His residue of lysozyme is photooxidized with a single kinetic. Two photooxidation kinetics were obtained for the Trp residues of both native proteins, an indication that in both cases there are Trp residues that are differently exposed to the solvent. The wavelengths of maximum fluorescent emission of the Trp residues were different for the 2 proteins, an effect which can also be explained in terms of a difference in the environment of these residues. The modified form of these proteins emit at wavelengths longer than those of the native forms. When modified the proteins photooxidize with noticeably greater quantum yields.
PHOTOCHEMICAL-LIKE BEHAVIOR OF RIBOFLAVIN IN THE DARK PROMOTED BY ENZYME-GENERATED TRIPLET ACETONE
(1988) ROJAS, J; SILVA, E
Triplet acetone obtained enzymically by the isobutyraldehyde/peroxidase/O2 system transfers its energy to the riboflavin, which changes into a decompsition product of lumichrome type and produces aggregates. When ribflavin and trypotphan are added to this system, further products.sbd.formylkynurenine and an adduct between the riboflavin and the tryptophan.sbd.are formed. The adduct can be separated by gel filtration and ion exchange chromatography and is similar to that prepared by irradiating riboflavin in the presence of tryptophan.
PHOTOINDUCED RIBOFLAVIN BINDING TO THE TRYPTOPHAN RESIDUES OF BOVINE AND HUMAN SERUM ALBUMINS
(1991) TAPIA, G; SILVA, E
The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of C-14-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of C-14-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.
PHOTOINTERACTION OF BENZOPHENONE TRIPLET WITH LYSOZYME
(1989) ENCINAS, MV; LISSI, EA; VASQUEZ, M; OLEA, AF; SILVA, E
The quenching of the benzophenone triplet by lysozyme and its constituent amino acids in aqueous solutions have been studied. Native lysozyme quenches the benzophenone triplet with a high rate constant, 4 .times. 109 M-1s-1. The quenching process takes place with production of significant amounts of free ketyl radicals, .PHI.ketyl = 0.56, but with a very low benzophenone consumption yield (0.022). The consumption yield is considerably smaller than that observed for the free amino acids. This difference can be explained in terms of a dominant back hydrogen transfer to the protein in the disproportionation of the free radicals produced. Reduced and carboxymethylated lysozyme shows a higher quenching rate (7.8 .times. 109 M-1s-1) and a larger benzophenone consumption yield (0.07). The deactivation of the benzophenone triplet by the native protein leads to its inactivation, with a quantum yield of 0.01. Tryptophan and arginine residues are destroyed with a quantum yield of 0.01. In the modified enzyme tryosine and methionine groups are also consumed.
PHOTOREACTIONS OF RIBOFLAVIN WITH SPERMINE AND THEIR ROLE IN TRYPTOPHAN PHOTOCONSUMPTION INDUCED BY RIBOFLAVIN
(ELSEVIER SCIENCE SA, 1993) SILVA, E; ALMARZA, C; BERNDT, D; LARRONDO, L; LISSI, E
The influence of spermine (Spr) on riboflavin (Rb) and riboflavin-tryptophan (Rb-Trp) solutions irradiated at 450 nm under aerobic and anaerobic conditions was studied. In both systems, it is shown that Spr interacts with 3Rb probably giving spermine-derived and flavin-derived radicals. In the presence of oxygen, modified Rb is regenerated with the simultaneous production of the active oxygen species O2.- and/or HO2.. The addition of Spr to the Rb-Trp system under aerobic conditions leads to a small protection of Trp consumption, although oxygen consumption is increased. This small protection arises as a consequence of competitive interaction of Trp and Spr with 3Rb. The competition of Spr and oxygen for 3Rb could also explain the autocatalytic oxygen consumption observed in the presence of the amine. Under anaerobic conditions Spr produces a larger protection of Trp consumption, which is compatible with the larger Rb triplet lifetime expected under these conditions.
RIBOFLAVIN SENSITIZED PHOTOOXIDATION OF TYROSINE
(VERLAG HANS HUBER, 1994) SILVA, E; GODOY, J
The oxygen reactive species generated during the photooxidation of tyrosine sensitized by riboflavin were studied and the products formed were isolated. It was found that this process occurs preferently through a type I mechanism, which involves the direct interaction between the excited state of the sensitizer and tyrosine. The formation of O-2.-, H2O2 and .OH was detected along the photooxidative process. By changing H2O by D2O it could be established that O-1(2) has no significant role in the photooxidation of tyrosine sensitized by riboflavin. When a previously irradiated solution of tyrosine was applied to a Sephadex G-15 column, four fractions could be separated corresponding to products having hydrodynamic volumes larger than those of the reactants. By the use of C-14-tyrosine the aminoacidic origin of the products could be determined; one of them was identified as bityrosine, according to its fluorescence properties and H-1-NMR spectrum.
RIBOFLAVIN STATUS AND PHOTOINDUCED RIBOFLAVIN BINDING TO THE PROTEINS OF THE RAT OCULAR LENS
(1988) SALIMHANNA, M; VALENZUELA, A; SILVA, E
The presence of radioactivity in lenses of rats injected intraperitoneally with a solution of tryptophan and 14C-riboflavin, irradiated during 48 hours and under N2 atmosphere, was detected. The excitation fluorescence spectrum (.lambda.emission = 520 nm) of the soluble lens fraction corresponds to the riboflavin decomposition product of lumichrome type. When 14C-riboflavin enriched lenses were exposed to visible light, a photo-induced binding between riboflavin and a water insoluble protein fraction of the lenses was observed. This finding may help to clarify the lens modifications during aging and in cataractogenesis.
STUDY OF A PHOTOINDUCED LYSOZYME-RIBOFLAVIN BOND
(1985) FERRER, I; SILVA, E
Irradiation of lysozyme in the presence of riboflavin results in the formation of a lysozyme-riboflavin adduct. Reduction and carboxymethylation of the 4 disulfide bonds as well as the chemical modification of the Tyr residues and the photochemical alteration of the His residue in lysozyme, do not affect the formation of the photo-induced lysozyme-riboflavin bond. When the lysozyme-riboflavin adduct was subjected to mild acid hydrolysis and ion exchange chromatography, the retention of a compound containing 14C-riboflavin was observed. Free 14C-riboflavin, on the contrary, is not retained by the column. The photo-oxidation of free Trp in the presence of 14C-riboflavin, gave a compound which bound to the ion exchange resin like the above-mentioned derivative. The photo-oxidation of the Trp residues in lysozyme and in peptides obtained from lysozyme showed very high quantum yields, and these values were directly related to the incorporation of 14C-riboflavin in these samples.
TOXIC EFFECT OF A PHOTOINDUCED TRYPTOPHAN-RIBOFLAVIN ADDUCT ON F9-TERATOCARCINOMA CELLS AND PREIMPLANTATION MOUSE EMBRYOS
(1988) SILVA, E; SALIMHANNA, M; BECKER, MI; DEIOANNES, A
Solutions containing L-tryptophan and riboflavin exposed to visible light, under N2 atmosphere, yield a tryptophan-riboflavin adduct, able to inhibit the growth of cultured F9 teratocarcinoma cells. This same effect was found in the presence of a mixture of the tryptophan photooxidation products and the adduct, when using solutions previously irradiated with visible light in an O2 atmosphere. A cytotoxic effect was also observed with embryos incubated in the presence of a tryptophan-riboflavin adduct, in the latter case necrosis and embryo development arrest occured.
TRYPTOPHAN-RIBOFLAVIN PHOTOINDUCED ADDUCT AND HEPATIC-DYSFUNCTION IN RATS
(1988) DONOSO, MDN; VALENZUELA, A; SILVA, E
VISIBLE-LIGHT EFFECTS ON TUMORAL CELLS IN A CULTURE-MEDIUM ENRICHED WITH TRYPTOPHAN AND RIBOFLAVIN
(1994) EDWARDS, AM; SILVA, E; JOFRE, B; BECKER, MI; DEIOANNES, AE
When NSO/2 myeloid cell line and teratocarcinoma F9 cells were irradiated in Dulbecco's modified Eagle medium enriched with tryptophan and riboflavin, toxic photoproducts for these tumoral cells were generated. The active participation of O-1(2) and .OH was established using specific scavengers and quenchers. A cytotoxic effect was also observed when unirradiated tumoral cells were incubated in a previously irradiated culture medium enriched with tryptophan and riboflavin. When irradiated medium was used alone, enriched only with tryptophan or only with riboflavin, no toxic effect was observed. The relevance of charge transfer processes between triplet riboflavin and tryptophan in the generation of cytotoxic photoproducts is discussed.

Browsing by Author "SILVA, E"

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