Photochemical properties of Yt base in aqueous solution.

Photoreactivity of Yt base [I] has been studied in aqueous solution [pH approximately 6] saturated with oxygen. Two photoproducts (II,III], resulting from irradiation at lambda = 253.7 nm and lambda greater than or equal to 290 nm, were isolated and their structures determined. The quantum yield for Yt base disappearance [zeta dis] is 0.002 [lambda = 313 nm]. It was shown that dye-sensitized photooxidation of Yt base in aqueous solution occurs according to a Type I mechanism, as well as with participation of singlet state oxygen. Quantum yields, fluorescence decay times and phosphorescence of Yt base have been also determined.     

Photosensitized formation of ascorbate radicals by chloroaluminum phthalocyanine tetrasulfonate: an electron spin resonance study.

The chloroaluminum phthalocyanine tetrasulfonate sensitized photooxidation of ascorbic acid to ascorbate radical (A.-) was followed by electron spin resonance (ESR) spectroscopy. In air saturated aqueous media, steady-state amounts of A.- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished--more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN3 and tryptophan) was studied, as was replacement of water by D2O and saturation with O2. The results are indicative of free radical production by direct reaction between ascorbate ion and sensitized phthalocyanine (a Type I mechanism) in competition with the (Type II) reaction of HA- with singlet oxygen, a reaction which does not produce ascorbate radical intermediates.     

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.     

血清胆红素的光敏氧化

竹红菌乙素是一种芘醌类的光敏剂.实验结果表明在可见光的照射下.它能加速血清胆红素的光氧化,氧化速率提高5倍以上.比较在不同溶剂中各种活性氧淬灭剂对胆红素光氧化的抑制作用,指出血清胆红素光敏氧化反应包括自由基氧化反应(Ⅰ型反应)和单态氧氧化反应(Ⅱ型反应)等多重机制.     

Photochemically induced transformation of adrenochrome to adrenochrome-melanin

The photochemical decomposition of adrenochrome in aqueous and deuterated solutions by visible light was investigated. From the spectroscopic study the disappearance constant k = 4.8 x 10(-5) s-1 as well as quenching constant kq = 2.5 x 10(-1) [s M-1] and isotope effect kD/kH = 2 for singlet oxygen mechanism have been calculated. A possible chemical mechanism for the observed transformation of adrenochrome to the melanin polymer is discussed including the formation of the reactive intermediate species like cytotoxic quinones.     

Light-induced binding of riboflavin to lysozyme

Summary The 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 has been studied. When H₂O was replaced by D₂O a great increase in the rate of photoinactivation of lysozyme was observed. This finding, together with the fact that photooxidation is inhibited by singlet oxygen quenchers like NaN₃, suggests that these reactions occur via a singlet oxygen mechanism.During the course of the studies of the riboflavin sensitized photoinactivation of lysozyme, it was found that riboflavin is strongly bound to the enzyme as a result of illumination. This finding would explain the higher quantum yield observed when riboflavin is used, although this dye is bleached during irradiation.     

A comparative kinetic study on the singlet molecular oxygen-mediated photoxidation of alpha- and beta-chymotrypsins.

Kinetic aspects of the sensitized photooxidation of alpha- and beta-chymotrypsins have been studied at pH 6 and 8. The sensitization, employing classical O2(1Deltag)-photogenerators, such as xanthene dyes, is a kinetically intricate process because of the presence of ground state dye-protein associations and to the simultaneous participation of superoxide ion and singlet molecular oxygen [O2(1Deltag)]. Both proteins, that possess the same distribution pattern of photooxidizable amino acids, suffer a pure O2(1Deltag)-mediated photodynamic attack, using the carbonylic sensitizer Perinaphthenone. Overall and reactive rate constants for the O2(1Deltag)-quenching (in the order of 108 and 107/M/s, respectively), and rates of oxygen consumption determined by time-resolved, spectroscopic and polarographic methods indicate that alpha- and beta-chymotrypsins are less photooxidizable at pH 6, as a result of an enhancement of the O2(1Deltag)-physical quenching component. In general terms, beta-chymotrypsin exhibits the greater overall proclivity to interact with O2(1Deltag), whereas structural factors, possibly evidenced by a higher exposure of the reactive tryptophan residues, impart an increased photooxidation degree to the proteins at pH 8, specially to the alpha-chymotrypsin.     

Photodynamic degradation of vitamin E induced by psoralens.

Psoralens and other furocoumarins currently used in PUVA photochemotherapy are shown to have, to a variable extent, the ability to hasten the rate of ultraviolet-induced photooxidation of alpha-tocopherol (alpha-T) in ethanol or ethanol-phosphate buffer (pH 6.8). The sensitizing effect varies significantly with the substrate concentration and the nature of the furocoumarin used, and is dependent on the presence of oxygen. Scavengers of singlet oxygen, e.g., sodium azide, markedly inhibit the psoralen-sensitized photooxidation of alpha-T, whereas superoxide dismutase exerts an opposite, accelerating effect on the reaction rate. Catalase has no significant influence on the kinetics of alpha-T decay. Analysis of the products formed by psoralen-sensitized photooxidation of alpha-T in ethanol-phosphate buffer showed the presence of alpha-tocopherolquinone, its 2,3-epoxide and two related compounds containing the 7-oxaspiro[4.5]dec-1-ene-3,6-dione ring system. The nature of these products, coupled with the results of the kinetic experiments, suggest that psoralens induce a type II, oxygen-dependent photodegradation of alpha-T primarily mediated by singlet oxygen.     

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.     

Oxygen consumption and diffusion effects in photodynamic therapy

Effects of oxygen consumption in photodynamic therapy (PDT) are considered theoretically and experimentally. A mathematical model of the Type II mechanism of photooxidation is used to compute estimates of the rate of therapy-dependent in vivo oxygen depletion resulting from reactions of singlet oxygen (1O2) with intracellular substrate. Calculations indicate that PDT carried out at incident light intensities of 50 mW/cm2 may consume 3O2 at rates as high as 6-9 microM s-1. An approximate model of oxygen diffusion shows that these consumption rates are large enough to decrease the radius of oxygenated cells around an isolated capillary. Thus, during photoirradiation, cells sufficiently remote from the capillary wall may reside at oxygen tensions that are low enough to preclude or minimize 1O2-mediated damage. This effect is more pronounced at higher power densities and accounts for an enhanced therapeutic response in tumors treated with 360 J/cm2 delivered at 50 mW/cm2 compared to the same light dose delivered at 200 mW/cm2. The analysis further suggests that the oxygen depletion could be partially overcome by fractionating the light delivery. In a transplanted mammary tumor model, a regimen of 30-s exposures followed by 30-s dark periods produced significantly longer delays in tumor growth when compared to the continuous delivery of the same total fluence.     

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.     

NADPH2 and organic hydroperoxide-dependent oxidation of adrenaline to adrenochromes in liver and brain microsomes

It was shown that oxidation of adrenaline to adrenochrome in microsomal membranes of the brain and liver in the presence of NADP . H2 or NAD . H2 is mainly accounted for by the formation of a superoxide anion radical. The formation of adrenochrome from adrenalin was found to depend on organic hydroperoxides (natural and synthetic). The organic hydroperoxide-dependent oxidation of adrenochrome involves singlet oxygen. In microsomal fractions of the liver the organic peroxide-dependent oxidation of adrenalin was catalyzed by cytochrome P-450.     

Psoralens sensitize glutathione photooxidation in vitro

In vitro experiments are reported showing that psoralens and other furocoumarins of current pharmacological interest, e.g., angelicin and 4,6,4'-trimethylangelicin, all have, to a variable extent, the ability to sensitize the photooxidation of glutathione in ethanol/phosphate buffer with pyrex-filtered ultraviolet light. Besides substrate concentration and the nature of the furocoumarin used, the rate of the sensitized reaction is markedly dependent on the partial pressure of oxygen and the pH of the medium, being progressively faster on passing from pH 5 to pH 8.5. Scavengers of superoxide ions (superoxide dismutase), hydrogen peroxide (catalase) and singlet oxygen (sodium azide, diazabicyclooctane, sorbic acid) have little or no inhibitory effect on the reaction rate. These and other data suggest that furocoumarins can directly sensitize glutathione photooxidation by forming a charge transfer complex which is driven to the oxidized products in the presence of oxygen. The possible relevance of these results to the mechanisms of skin melanin hyperpigmentation induced by furocoumarins and ultraviolet light is discussed.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Detection of singlet oxygen and its role in dye-sensitized photooxidation in aqueous and micellar solutions

Indirect methods for the detection of singlet oxygen in dye-sensitized photooxidation based on its interception by some singlet oxygen acceptors in aqueous and micellar solutions are discussed. Mechanistic aspects and some applications of a very sensitive method using p-nitrosodimethylaniline in the presence of imidazole (RNO + imidazole method) are also treated. The technique of competition kinetics with a singlet oxygen quencher N-3 which can serve for the determination of the role of singlet oxygen is discussed as well. Such competition with tryptophan and guanosine shows that these substrates react exclusively or predominantly via the singlet oxygen mechanism in the presence of hematoporphyrin as sensitizing dye.     

Mechanisms involved in the chemical inhibition of the eosin-sensitized photooxidation of trypsin

A large series of compounds was screened for ability to protect trypsin from eosin-sensitized photodynamic inactivation. Eosin-sensitized photooxidation reactions of this type typically proceed via the triplet state of the dye and often involve singlet state oxygen as the oxidizing entity. In order to determine the mechanisms by which trypsin is protected from photoinactivation, a number of good protective agents (inhibitors) and some non-protective agents were selected for more detailed flash photolysis studies. Good inhibitors such as p-phenylenediamine, n-propyl gallate, serotonin creatinine sulfate and p-toluenediamine competed efficiently with oxygen and with trypsin for reaction with the triplet state of eosin. The inhibitors were shown to quench triplet eosin to the ground state and/or reduce triplet eosin to form the semireduced eosin radical and an oxidized form of the inhibitor. In the latter case, oxidized inhibitor could react by a reverse electron transfer reaction with the semi-reduced eosin radical to regenerate ground state eosin and the inhibitor. The good inhibitors also competed effectively with trypsin for oxidation by semioxidized eosin, thus giving another possible protective mechanism. Non-inhibitors such as halogen ions and the paramagnetic ions Co++, Cu++ and Mn++ reacted only slowly with triplet and with seimioxidized eosin. The primary pathway for the eosin-sensitized photooxidation of trypsin at pH 8.0 involved singlet oxygen, although semioxidized eosin may also participate.     

Photochemiluminescence arising during sensitized photooxidation of trypsin solutions

Kinetic study of eosin-sensitized photochemoluminescence (PCL) in tripsin solutions was carried out. Kinetics of luminescence increase and decrease, connection between sensitized PCL with triptophane photodissociation in protein were investigated. Effect of the concentrations of protein and oxygen in solution on the parameters of eosin-sensitized PCL was studied in order to establish the succession of processes resulting in the formation of free radicals. The experimental data made it possible to propose a scheme of primary processes responsible for PCL, which shows that the formation of protein radicals proceeded in the reactions between the triplet excited states of dye and singlet oxygen and protein triptophyle residues. It was found that during the formation of radicals in the air at small concentrations of proteins singlet oxygen played a major role in the sensitized photooxidation of protein. It was shown that the mechanism of processes responsible for sensitized PCL, determining postluminescence is similar to tripsin chemoluminescence under UV-illumination. Some kinetic parameters of the processes proceeding during sensitized photooxidation were determined and estimated.     

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

Summary The effect of the substitution pattern on the kinetics of the Type II (O₂(¹_g)-mediated) dye-sensitized photooxidation of a series of nine tyrosine derivatives was investigated. Overall (k_t) and reactive (k_r) 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_g) 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.Abbreviations O ₂(³ _g ^– ) ground state triplet oxygen - O ₂(¹_g) singlet molecular oxygen - Tyr L tyrosine - TyrD tyrosine derivatives - Eos eosine - RB rose bengal - FFA furfuryl alcohol - DMA 9, 10-dimethyl anthracene     

Photooxidation products of 7,12-dimethylbenz[a]anthracene.

The principal products of the photooxidation of 7,12-dimethylbenz[a]-anthracene (DMBA) in aqueous solutions by photooxidation induced by laboratory lighting have been characterized by high performance liquid chromatograms (HPLC), ultraviolet and mass spectrograms and by comparisons with authentic samples. The products identified were the 7,12-epidioxy-7,12-dihydro-7-12-dimethyl-, 7,12-dione, 7-hydroxymethyl-12-methyl-, 12-hydroxymethyl-7-methyl-, 7-formyl-12-methyl-, 12-formyl-7-methyl-, and 12-hydroxy-12-methyl-7-one derivatives of benz[a]-anthracene. The HPLC profile of products is similar to that obtained from oxidation of DMBA by 'one-electron' reagents, singlet oxygen, or liver microsomal metabolism. The first points of attack are the 7- and 12- positions. The mechanism of photooxidation appears to be generation of singlet oxygen by photodynamic effect of DMBA. None of the products is photosensitizing, however.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.