Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).

Sensitized photooxidation of Di- and tripeptides of tyrosine.

This paper studies the dye-sensitized photooxidation of tyrosine (tyr) and tyr di- and tripeptides (tyr-tyr and tyr-tyr-tyr) mediated by singlet molecular oxygen (O2[1 delta g]) in alkaline media. Photooxidation quantum efficiencies (phi r) were obtained by determining the overall and reactive rate constants of interaction with the oxidative species, employing the time-resolved O2(1 delta g) phosphorescence detection method and static-photolysis actinometric method, respectively. The interaction of O2(1 delta g)-tyr derivatives occurs through an intermediate encounter complex with polar character. Ionization of the phenolic OH group of tyr derivatives and the polarity of the solvent favors the overall interaction. Nevertheless, phi r values decrease when changing from water to MeCN-water medium. This indicates that the reactive deactivation of the encounter complex, probably an entropy-controlled step, may be affected by solvent polarity in the same way as those processes in which charges are neutralized along the reaction pathway. Photooxidation quantum efficiencies indicate that the contribution to O2(1 delta g) physical quenching (a second alternative deactivation route for the encountered complex [O2(1 delta g)-tyr derivatives]) increases with the complexity of the peptide. As a result, the selfprotection of the peptidic entity against physical quenching also increases. The information obtained from the fractional consumption mol O2/mol tyr derivative (in tyr, the di- and tripeptides and the respective methyl ester of tyr and the tripeptide), together with the evolution (either consumption and/or generation) of primary amino groups upon photosensitized irradiation of the same compounds clearly indicates that the photooxidation of di- and tri-tyr peptides proceeds with the breakage of peptidic bonds. As a consequence, in the final balance each tyr unity behaves as an independent photooxidizable target.

Singlet molecular oxygen-mediated photo-oxidation of tetracyclines: kinetics, mechanism and microbiological implications.

Members of the biologically active series tetracyclines (TCs) suffer visible light-promoted photodynamic degradation to different extents, depending on their respective chemical structures and reaction conditions (solvent polarity and pH). The photo-oxidation is accompanied by a partial loss of the antimicrobial power. The photodamage is very fast in the alkaline pH range and less aggressive. although not negligible in kinetic terms, in the physiological pH region. Photo-oxidation quantum efficiencies, evaluated for eight TC derivatives, through singlet molecular oxygen [O2(1Delta(g))] phosphorescence detection, spectrophotometric and polarographic methods, range from 0.12 to 0.65 as upper limits in alkaline medium. The photo-oxidation essentially proceeds via a O2(1Delta(g)) mediated process, with rose bengal or eosine as dye-sensitizers, Nevertheless, as a minor reactive pathway,the excited triplet state of the dye sensitizers interacts with TCS in a competitive process with O2(1Delta(g) generation. The O2(1Delta(g)-mediated photo-oxidation of TCs appears to be a plausible mechanism to account for their phototransformations in biological media, in the presence of visible-absorbing pigments. In both highly and moderately polar media, the quenching of the excited oxygen species is mainly represented by a reactive interaction. It is exerted by the TC molecule through a cooperative effect from the different contributions of several nuclear and extranuclear O2(1Delta(g)-sensitive substituents, as discussed in detail in this paper. The TC lower than 0.03 in the most favourable cases. Nevertheless, the TC photoproduct, formed through direct irradiation, efficiently generates O2(1Delta(g) with Phi(Delta)=0.24. This important finding constitutes the first direct evidence of Type II sensitization by TC photoproducts, and could contribute to the elucidation of the mechanism of TC phototoxicity.

Influence of the pH on the photodynamic effect in lysozyme A comparative kinetic study with the sensitized photooxidation of isolated amino acids.

The kinetics of the eosin-sensitized photooxidation ([O2((1)Δg)]-mediated) of the protein lysozyme (Lyso) was investigated under two different pH conditions (pH 7 and pH 11). Rates of oxygen consumption and the fade in the protein fluorescence spectrum upon sensitized irradiation were monitored. Parallel studies on both denatured Lyso (absence of the four-S-S- bridges in the protein) and different mixtures of the photooxidizable amino acids of Lyso were also carried out. The mixtures maintained the same molar ratio as in the native protein, and were selected just in order to throw into relief the preferential amino acids that were being photooxidized at both pH values.Under work conditions Lyso was only photooxidizable at pH 7, whereas the opposite accounted for the denatured protein: only measurable oxygen consumption was detected at pH 11. Nevertheless, Lyso at pH 11, evidenced an important physical quenching of O2((1)Δg) due to the Tyr and Trp residues.The results for the native protein were interpreted on the basis of a previously described dark complex Eosin-Lyso, which selectively favours the photooxidation of the bounded protein. The Trp residues were the main reactive entities in the native protein. The photodinamic effect in denatured Lyso was characterized by the prevalence of Tyr residues as photooxidizable targets.In the discussion of the results, a comparisson with the photooxidation kinetics of the mixtures of free amino acids was made.

Influence of the nuclear and extranuclear substitution on the singlet molecular oxygen [O 2( (1)Δ g)]-mediated photooxidation of tyrosine derivatives: A kinetic study.

The effect of the substitution pattern on the kinetics of the Type II (O2((1)Δg)-mediated) dye-sensitized photooxidation of a series of nine tyrosine derivatives was investigated. Overall (kt) and reactive (kr) rate constants for the interaction of the excited oxygen species with the amino acid derivatives were determined. A parallel study on solvent and pH effects was carried out.The presence of different substituents in nuclear positions or in the amino acid side chain greatly affect the photooxidation rates.An upper limit for photooxidation quantum yield, calculated from the kinetic data, varies from 0.03 to 0.25, being the higher for halogenated tyrosines and the lower for esterified tyrosines and for the nitro-derivative.The variation of solvent polarity and pH of the reaction medium confirm that the presence of the ionized phenolate group in tyrosine, clearly dominates the quenching process. As already postulated for generic phenolic derivatives, it proceeds through a polar intermediate complex which posses some component of charge-transfer character.Esterification of the carboxilic acid of tyrosine selectively decreases the contribution of the reactive step to the overall process of O2((1)Δg) quenching. An amide group in the same position does not produce noticiable changes in this sense. The presence of a highly deactivating nitro group in nuclear positions greatly diminishes the magnitude of both overall and reactive interactions.For all three, o-, m- and p-tyrosine the values of photooxidation quantum yields show an excellent parallelism with the rates of consumption of the - NH2 group of the amino acid chain, upon sensitized irradiation. It could react, in the cases of 0- and m-tyrosine in a secondary, non photochemical, step.