The carbonate radical: its reactivity with oxygen, ammonia, amino acids, and melanins.

The carbonate radical (CO 3 (*-)) is of importance in biology and chemistry. We used pulse radiolysis to generate the CO 3 (*-) radical and show there is no reaction with oxygen. However, in the presence of ammonia the CO 3 (*-) radical is removed by NO (*), which itself arises from the scavenging of NH 2 (*) by oxygen, and the mechanism of this process is reported. The CO 3 (*-) radical shows complex decay patterns in the presence of ammonia, which can be understood as a balance between the radical-radical reaction CO 3 (*-) + CO 3 (*-) and CO 3 (*-) + NH 2 (*) (the amino radical). Also, we report reactivity with glycine and alanine and with melanin models. The CO 3 (*-) reacts with both dopa-melanin (DM, a model of black eumelanin) and with cysteinyl-dopa-melanin (CDM, a model of red/blond phaeomelanin). However, the reaction rate constant is much higher with CDM than with DM.

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides.

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.

A pulse radiolysis investigation of the oxidation of methoxylated metabolites of indolic melanin precursors.

The rate constants associated with the series of successive transient absorptions initiated by one-electron oxidation of 6-hydroxy-5-methoxyindole (6H5MI) and its isomer 5-hydroxy-6-methoxyindole (5H6MI) have been studied by pulse radiolysis. These close analogues of 5,6-dihydroxyindole (DHI) are metabolites of the oxidative melanogenic pathway. The species initially produced from N3. oxidation of both methoxyindoles at pH 7.2-7.4 are assigned as the corresponding semiquinones. That from 6H5MI shows peak at 500, 370 and 330 nm, very close to those of the semiquinone of DHI, whereas the semiquinone of 5H6MI shows no absorption at 500 nm but bands at 420 and 340 nm. These spectral differences are attributed to marked changes in the degrees of electron delocalisation for the two types of radical, both rings of the indole being involved for the 6H5MI radical but only the benzenoid moiety for the 5H6MI radical. In both cases, the radicals decayed, probably by disproportionation, into products which absorbed in the 400-420 nm region. For 6H5MI, the subsequent decay in this region was best fitted by two consecutive first-order processes which were both strongly base-catalysed. The first of these processes is assigned to partial decay via deprotonation of the corresponding quinonoid cation to form an equilibrium mixture of this cation and the corresponding quinone methide. The second process is assigned to reaction of the quinone methide with water yielding hydroxylated product(s) which may subsequently react with remaining quinonoid cation or quinone methide to give dimeric product(s) with broad absorption centreing in the 550 nm region detected 0.5 s after the pulse. For 5H6MI, the decay at 430 nm fitted a single first-order process, which was weakly base-catalysed. This process is attributed to deprotonation of the corresponding quinonoid cation to the corresponding quinone imine absorbing below 350 nm, which was stable for at least tens of seconds. The current experiments suggest that our previous analogues observations (Lambert et al. (1989) Biochim. Biophys. Acta 993, 12-20) on the oxidation of the melanogenic precursors DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) may be interpreted, as with 6H5MI, in terms of the corresponding indolequinones decaying into equilibrium mixtures of quinone, quinone imine and quinone methide. These decay via reaction of the methide with water generating hydroxylated species which proceed to give the coloured product(s) absorbing in the 550 nm region.

Interaction of radicals from water radiolysis with melanin.

Melanins are considered to be natural photoprotectors in the melanocytes and keratinocytes of the skin. These pigments have also been suggested to play an important role in protection of melanin-containing cells against ionising radiation. Various mechanisms have been proposed to explain the protective role of melanin which invoke the radical scavenging properties of the polymer. In the present work the reactions of melanins with radicals generated in aqueous media by pulse radiolysis have been studied. Time-resolved changes in absorbance of the melanin or the radical species were recorded at selected wavelengths. Experiments were carried out on synthetic dopa- and 5-S-cysteinyldopa-melanins and a natural melanin in phosphate buffer (pH 7.4). Under the conditions employed, melanin reacted predominantly with either oxidising (OH., N3.) or reducing (eaq-, CO2-) species. We were also able to monitor the interaction of melanin with superoxide radical, which was reducing in this case. Detailed analysis of transient changes in melanin absorbance, detected at different wavelengths, was demonstrated to be a convenient method for studying redox processes of this substance, as shown by model experiments using ferricyanide and dithionite as oxidising and reducing agents, respectively. Among the radicals studied, OH. exhibited the strongest reactivity with melanins. Apparent rate constants for the reactions of radicals with autoxidative dopa-melanin (1.5 X 10(9) M-1 X s-1, 2.6 X 10(8) M-1 X s-1, 1.8 X 10(8) M-1 X s-1, 5 X 10(5) M-1 X s-1, 10(6)-10(7) M-1 X s-1 for OH., eaq-, N.3. O2- and CO2-, respectively) are reported. The reactivity of melanins with radicals from water radiolysis and their effect on pigment properties are discussed in terms of the structure and possible biological role of the pigments.

Triplet state characteristics and singlet oxygen generation properties of anthracyclines.

The triplet states of adriamycin (Ad), daunomycin (D) and two daunomycin analogues, daunomycinone (Dc) and daunomycin N-trifluoroacetamide (DAc), have been studied using laser flash photolysis and pulse radiolysis techniques. Triplet lifetimes, molar absorption coefficients, energy levels and quantum yields have been obtained for Dc and DAc, and estimated for D and Ad. Time-resolved near-infrared singlet oxygen luminescence measurements have been carried out on D, Ad and 5-iminodaunomycin (5-ID) in 2H2O solution and Dc in benzene solution at room temperature. Singlet oxygen quenching by the water-soluble anthracyclines was observed and a second-order rate constant of approx. 10(8) M-1.s-1 obtained. Electron spin resonance experiments have demonstrated that D photoexcited at lambda less than or 365 nm gives rise to singlet oxygen as shown by its reaction with 2,2,6,6-tetramethyl-4-piperidone to give the corresponding nitroxyl radical. Although all the anthracyclines studied have the ability to photosensitize the formation of singlet oxygen, the quantum yields are very low (phi delta approximately 0.02-0.03), suggesting that these anthracyclines would be poor photodynamic sensitisers.

Effects of the micro-environment on the photophysical properties of hematoporphyrin.

The photochemical degradation of histidine, cysteine and tyrosine with hematoporphyrin as sensitizer was potentiated by the presence of Sephadex, BioGel or Percoll particles. This effect could only partly be explained by binding of the sensitizer to the gel particles, leading a.o. to monomerization of aggregated sensitizer molecules in the aqueous environment. The hematoporphyrin triplet state life time increased from 250 microseconds in phosphate buffer to 1992 microseconds in the presence of 50% Percoll. Most likely the effect of the gel particles on the sensitizer triplet state is, at least partly, mediated by the solvent. A plausible explanation seems to be that the vicinal water structure at the particle interface stabilizes the sensitizer triplet.

A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas.

The unstable quinones of 3,4-dihydroxyphenylalanine (dopa) and the most abundant cysteinyldopa isomers (2S-, 5S- and 2,5S,S'-) have been generated rapidly via disproportionation of their respective semiquinones prepared pulse radiolytically by one-electron oxidation of the corresponding dopas with azide radicals. Dopaquinone decays via a base-catalysed unimolecular cyclisation yielding leucodopachrome which, under the present conditions, is immediately oxidised by remaining dopaquinone to form dopachrome and dopa back again. Addition of cysteine increased the rate of dopaquinone decay and precluded dopachrome formation. By contrast, the cysteinyldopa quinones decayed via an acid-catalysed unimolecular cyclisation involving the cysteine side chain to form a cyclic quinone-imine observed directly for the first time. These quinone-imine intermediates subsequently rearranged to more stable phenolic benzothiazine isomers. The addition of cysteine had little effect on cysteinyldopa quinone decay and did not prevent quinone-imine formation. The absorption spectra, extinction coefficients and rate constants for formation and decay of these various transient species involved in melanisation are reported.

A pulse radiolysis investigation of the oxidation of indolic melanin precursors: evidence for indolequinones and subsequent intermediates.

The rate constants associated with the series of successive transient absorptions initiated by one-electron oxidation of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), precursors of melanin, and N-methyl-5,6-dihydroxyindole (NMDHI), a model compound, have been studied by pulse radiolysis. The initial transient species resulting from N3. oxidation reaction at pH 7.3-7.4 are assigned as the corresponding semiquinones. In each case, these radicals decayed, probably by disproportionation, into products most readily monitored in the 400-430 nm region. For DHI, the decay in this region could be fitted by two parent concentration independent first-order processes. These may correspond to transformations between 5,6-indolequinone, and its quinone-imine and quinone-methide tautomers. With NMDHI, on the other hand, a single longer-lived product with a peak around 430 nm predominated after decay of the corresponding radical, due almost certainly to N-methyl-5,6-indolequinone. The data appear to exclude significant melanin polymerisation by condensation of semiquinones, reaction of semiquinones with dihydroxyindoles, self-addition of indolequinones or tautomers, or reaction of indolequinones or tautomers with the parent dihydroxyindoles. It is suggested that polymerisation of melanin may rather occur by stepwise addition of indolequinone methide/imine to reduced oligomeric species.

Photochemical, photophysical and photobiological properties of some methylallopsoralens which are potential agents for photochemotherapy.

Two new trimethylallopsoralens, 4,7,4'- and 4,7,5'-trimethylallopsoralen, form molecular complexes with DNA and by successive UVA (320-400 nm) irradiation photobind monofunctionally to the macromolecule. The DNA photobinding rates at 365 nm and photobinding quantum yields at 330 nm are markedly higher for 4,7,4'-trimethylallopsoralen than for 4,7,5'-trimethylallopsoralen. Their capacities to generate singlet oxygen in water and benzene are low, particularly for 4,7,4'-trimethylallopsoralen, and the two trimethylallopsoralens completely lack skin phototoxicity on guinea-pig skin. Both compounds show antiproliferative activity in terms of DNA synthesis inhibition in Ehrlich cells and T2 phage infectivity higher than that displayed by angelicin. In view of its monofunctional character, lack of skin phototoxicity, low singlet oxygen yield and antiproliferative activity, 4,7,4'-trimethylallopsoralen deserves further clinical studies as a potential photochemotherapeutic agent.

Reactivity of butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) is a synthetic antioxidant that is widely used as an additive in foodstuffs to prevent spoiling. The physical-chemical properties of BHT and many related phenols have been examined previously although the mechanisms by which it exerts its antioxidant properties are poorly understood. The reactivity of BHT with singlet oxygen [O2(1 delta g)] and a number of radical species has been examined using the techniques of time resolved luminescence and pulse radiolysis. In benzene solution BHT reacted with O2(1 delta g) at a bimolecular rate constant of 1.3 x 10(6)M-1s-1. The one-electron oxidized, phenoxyl type BHT radical was generated using pulse radiolysis and the absorption spectrum showed a maximum at 400 nm. BHT reacts slowly with many radical species and upper limits for the bimolecular rate constant for reaction with several electron transfer processes are presented. The antioxidant role of BHT is discussed in terms of its reactivity, localization, and stability.

Beta-carotene with vitamins E and C offers synergistic cell protection against NOx.

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for beta-carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti-oxidants and this is explained in terms of an electron transfer reaction in which the beta-carotene radical is repaired by vitamin C.

Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals.

The human skin and eye melanin is commonly viewed as an efficient photoprotective agent. To elucidate the molecular mechanism of the melanin-dependent photoprotection, we studied the interaction of two synthetic melanins, dopa-melanin and cysteinyldopa-melanin, with a wide range of oxidising and reducing free radicals using the pulse radiolysis technique. We have found that although both types of free radicals could efficiently interact with the synthetic melanins, their radical scavenging properties depended, in a complex way, on the redox potential, the electric charge and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the polymer redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidised groups accessible to reaction with the radicals. Although with many radicals studied, melanin interacted via simple one-electron transfer processes, the reaction of both melanins with the strongly oxidising peroxyl radical from carbon tetrachloride, involved radical addition. Our study suggests that the free radical scavenging properties of melanin may be important in the protection of melanotic cells against free radical damage, particularly if the reactive radicals are generated in close proximity to the pigment granules.

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments.

The one-electron reduction potentials of the radical cations of five dietary carotenoids (beta-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980-1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.

The reduction potential of the beta-carotene.+ /beta-carotene couple in an aqueous micro-heterogeneous environment.

There is a resurgence of interest in the role of electron transfer reactions involving beta-carotene in photosynthesis. There is also current debate on the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations such as smokers. The impact of dietary carotenoids on health may well be also related to radical reactions. A key parameter in biological systems is therefore the one-electron reduction potential of the carotenoid radical cation, now reported for the first time in a model biological aqueous environment. The value obtained is 1.06+/-0. 01 V and is sufficiently high to oxidise cell membrane proteins, but is low enough to repair P(680).+ in the photosynthetic reaction centre.

Interaction of melanin with carbon- and oxygen-centered radicals from methanol and ethanol.

The interaction of dopa-melanin (DM) and cysteinyldopa-melanin (CDM) with carbon- and oxygen-centered radicals generated by benzophenone-photosensitized hydrogen abstraction from ethanol, or by pulse radiolysis of aqueous solutions of methanol and ethanol, is reported. Photosensitized formation of carbon-centered radicals and their interaction with melanin was monitored by electron paramagnetic resonance (EPR) spin trapping using DMPO, and via the melanin free radical signal itself. In the pulse radiolysis experiments, the interaction of DM or CDM with hydroxymethyl, hydroxyethyl, and the corresponding methanol peroxyl radical was monitored by recording time-dependent changes of the melanin absorbance at selected wavelengths. The data indicate that both melanins are good scavengers of carbon-centered radicals, with corresponding rate constants in the range of 10(7) to 10(8) M-1 s-1. Significantly, compared to DM, CDM is also an exceptionally efficient scavenger of oxygen-centered radicals derived from methanol with corresponding rate constants of 2.7 x 10(4) and 2 x 10(6) M-1 s-1 for DM and CDM, respectively. The results are discussed with reference to the potential role of melanin in protecting the integrity of melanosomes by inhibiting peroxidation of lipid components of the organelle membrane.

Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells.

Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.

Blue light-induced singlet oxygen generation by retinal lipofuscin in non-polar media.

Accumulation of lipofuscin (LF) is a prominent feature of aging in the human retinal pigment epithelium (RPE) cells. This age pigment exhibits substantial photoreactivity, which may increase the risk of retinal photodamage and contribute to age-related maculopathy. In a previous study, we detected singlet oxygen generation by lipofuscin granules excited with blue light. In this paper we investigated the ability of hydrophobic components of lipofuscin to photogenerate singlet oxygen in non-polar environments. Singlet oxygen was detected directly by monitoring its characteristic phosphorescence at ca 1270 nm. The action spectrum of singlet oxygen formation indicated that this process was strongly wavelength-dependent and its efficiency decreased with increasing wavelength by a factor of ten, comparing 420 nm and 520 nm. The quantum yield of singlet oxygen increased with increasing concentration of oxygen. Using laser flash photolysis we studied the possible mechanism of singlet oxygen formation. The observed transient, with a broad absorption spectrum peaking at around 440 nm, was identified as a triplet with lifetime ca 11 microseconds. It was quenched by both molecular oxygen and beta-carotene with concomitant formation of a beta-carotene triplet state. These results indicate the potential role of hydrophobic components of lipofuscin in blue light-induced damage to the RPE.

Influence of rhodamine 123 on the photosensitizing properties of porphyrins.

The photophysical and photochemical properties of porphyrins were profoundly changed upon addition of rhodamine 123. The Soret band of the porphyrins shifted to higher wavelengths, the fluorescence yield of the porphyrins decreased with unaltered decay rates, and their triplet state was quenched. These observations indicate a strong interaction between porphyrins and rhodamine 123 and formation of 1:1 nonfluorescent complexes, of which the binding constants were determined. Illumination of a porphyrin in the presence of rhodamine 123 resulted in the formation of a porphyrin radical cation, which could be detected with ESR spectroscopy. Quenching of the triplet state of the porphyrins by rhodamine 123 resulted in a decreased singlet oxygen yield and a decrease of the photooxidation of histidine, methionine, tyrosine, and tryptophan. However, the oxidation of thiol compounds was increased and the stoichiometry of the reaction between cysteine and oxygen changed from 2 to 3.8 mol cysteine/ mol oxygen. These results show that the presence of rhodamine 123 converted the for porphyrins prevalent energy transfer (type II) reaction to an electron transfer (type I) reaction.