Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.     

Interactions of dietary carotenoids with singlet oxygen (1O2) and free radicals: potential effects for human health

The dietary carotenoids provide photoprotection to photosynthetic organisms, the eye and the skin. The protection mechanisms involve both quenching of singlet oxygen and of damaging free radicals. The mechanisms for singlet oxygen quenching and protection against free radicals are quite different - indeed, under some conditions, quenching of free radicals can lead to a switch from a beneficial anti-oxidant process to damaging pro-oxidative situation. Furthermore, while skin protection involves β-carotene or lycopene from a tomato-rich diet, protection of the macula involves the hydroxyl-carotenoids (xanthophylls) zeaxanthin and lutein. Time resolved studies of singlet oxygen and free radicals and their interaction with carotenoids via pulsed laser and fast electron spectroscopy (pulse radiolysis) and the possible involvement of amino acids are discussed and used to (1) speculate on the anti- and pro-oxidative mechanisms, (2) determine the most efficient singlet oxygen quencher and (3) demonstrate the benefits to photoprotection of the eye from the xanthophylls rather than from hydrocarbon carotenoids such as β-carotene.     

Vascular effects of singlet oxygen (1O2) generated by photo-excitation on adrenergic neurotransmission in isolated rabbit mesenteric vein

It has been suggested that reactive oxygen species (ROS) may be involved in the regulation of vascular tone. However, the nature of ROS effects on vascular sensitivity remains to be elucidated. The present study was designed to investigate the effects of ROS, especially 1O2, on neurotransmission at the sympathetic neurovascular junction. Basal noradrenaline (NA) release, release of NA induced by electrical stimulation (ES), and resting NA release at the sympathetic nerve terminals were determined using a superfusion technique. The amount of NA was determined by HPLC; isometric tension changes evoked by ES were also recorded simultaneously. 1O2 was generated from Rose Bengal by photo-activation. The generation of 1O2 in the superfusate was monitored by electron spin resonance (ESR) using the spin trap 2,2,6,6-tetramethyl-4-piperidinol throughout the experimental time course. The ESR results confirmed that 1O2 was generated by photo-activation of Rose Bengal via the formation of 2,2,6,6-tetramethyl-4-hydroxyl-piperidinyloxy. Exposure of helical strips of rabbit mesenteric vein to 1O2 induced a significant increase in tension and NA release during the basal period, but had no effect on ES-induced release. L-histidine, an 1O2 scavenger, significantly inhibited the observed effects on vascular tension and NA release in response to 1O2. These results suggest that 1O2 may induce NA-mediated vasoconstriction at the postjunctional site, and may be associated with Ca(2+)-independent NA release from the prejunctional site of adrenergic neurotransmission.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

(Na+ + K+)-ATPase: inactivation and degradation induced by oxygen radicals.

1. Purified (Na+ + K+)-ATPase was irreversibly inhibited upon exposure to hydrogen peroxide, the superoxide anion, and the hydroxyl radical. 2. Comparison of the SDS-gel electrophoretic patterns of the ATPase samples exposed to these oxidants revealed that inhibition occurred either without gross structural changes, or concomitant with fragmentation and cross-linking of the enzyme subunits. 3. The oxidant modified ATPase was also shown to be more susceptible to degradation by several proteolytic enzymes.     

Photogeneration of free radicals (*OH and HB*-) and singlet oxygen (1O2) by hypocrellin B in TX-100 micelles microsurroundings

To solve the problems faced in clinical use of hypocrellins, a water-soluble preparation of Hypocrellin B (HB), HB-Triton X-100 (TX-100) micelles, was prepared. To evaluate the photodynamic activity, the free radicals (^•OH and HB^•¯) and singlet oxygen (

1

O

2

) generated via photosensitization of the preparation in aqueous solution were detected by using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was observed that

1

O

2

was formed with a quantum yield of 0.72, similar to that for HB in organic solvents, further, hydroxyl radicals (

•

OH) could also be efficiently produced by the new preparation, which have never before been detected following HB photoactivities. In addition, the semiquinone anion radicals (HB^•-) could also be generated via the self-electron transfer between an excited triplet state and a ground state molecule. The accumulation of HB^•- would replace that of

•

OH or

1

O

2

with the depletion of oxygen in the system. All these findings suggested that the HB-TX-100 micelles could play the photodynamic action through not only the type I mechanism by free radicals (^•OH, O₂^•- and HB^•-) but also the type II mechanism by singlet oxygen (

1

O

2

). It can be concluded further that the new preparation basically maintains the inherent photodynamic activity of HB, or even higher.     

ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

Sensitized photooxygenation and peroxidase-catalyzed inactivation of xanthine oxidase--evidence of cysteine damage by singlet oxygen

Xanthine oxidase (XO) has been investigated for its decreased activity in several cancerous tissues and constitutive generation of reactive oxygen species (ROS) in vivo seems to contribute significantly to its inactivation. Singlet oxygen (1O2) production has been suggested to be relevant when considering folic acid metabolism by cancer cells. Thus, the susceptibility of XO to inactivation by 1O2 generated either by the bioenergized systems folic acid/peroxidase/GSH/Mn2+/O2 and malonaldehyde/peroxidase/Mn2+/O2 or by methylene blue (MB) or eosin-sensitized photooxygenation was studied. Our results showed that other ROS were also responsible for XO inactivation when MB was used. In contrast, eosin produced almost exclusively 1O2. Kinetic studies of XO oxidation in the malonaldehyde/peroxidase system showed that histidine (His) is a competitive inhibitor with respect to XO. A similar result was observed in the eosin-photosensitized process, suggesting the involvement of 1O2 in both processes. In addition, an efficient quenching of XO oxidation by guanosine in the folic acid/peroxidase system was observed. Amino acid analysis revealed that cysteine (Cys) is more affected than other XO amino acids also prone to oxidation such as tyrosine (Tyr), methionine (Met) and His. These results indicate that 1O2 may cause oxidative damage to the Cys residues of XO, with loss of enzyme activity. Alteration of the flavin prosthetic site is hypothesized.     

Mechanism of versatile peroxidase inactivation by Ca(2+) depletion

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.     

Singlet oxygen ((1)Delta(g)O(2)) as the principal oxidant in myeloperoxidase-mediated bacterial killing in neutrophil phagosome.

Intraphagosomal viability of wild type E. coli and lycopene (a powerful (1)O(2) quencher)-producing transformant E. coli was investigated using human polymorphonuclear leukocytes as the cells for phagocytosis of opsonized viable bacteria. While the viability of both wild type and the transformant E. coli decreased very rapidly in the phagosome, but the viability of the lycopene-transformant in phagosomes was about 1.7 times higher than that of wild type E. coli after 5 min of incubation. The results were very similar to the results obtained when E. coli strains were exposed to (1)O(2) generated in myeloperoxidase-H(2)O(2)-Br(-) system (a pure (1)O(2) generating system) at pH 4.5. The reason for HOCl, which may be generated in the myeloperoxidase-H(2)O(2)-Cl(-) system under physiological conditions but does not become involved in bactericidal action, could be explained by the near neutral pH in phagosomes at which bacterial killing by chlorination is extensively attenuated. This is the first report which proved (1)O(2)-mediated bacterial killing in neutrophil-bacterial phagosomal system.     

Spectroscopic investigations of Er(3+) :CdO-Bi(2) O(3) -B(2) O(3) glasses

This article reports on the optical properties of Er³⁺ ions doped CdO–Bi₂O₃–B₂O₃ (CdBiB) glasses. The materials were characterized by optical absorption and emission spectra. By using Judd–Ofelt theory, the intensity parameters Ω_λ (λ = 2, 4, 6) and also oscillatory strengths were calculated from the absorption spectra. The results were used to compute the radiative properties of Er³⁺:CdBiB glasses. The concentration quenching and energy transfer from Yb³⁺–Er³⁺ were explained. The stimulated emission cross‐section, full width at half maximum (FWHM) and FWHM × values are also calculated for all the Er³⁺:CdBiB glasses. Copyright © 2011 John Wiley & Sons, Ltd.     

Membrane pores: a computer simulation of interacting pores analyzed by g1(tau) and g2(tau) correlation functions

Ion channels in a cell membrane were modeled by a computer simulation of fluctuating pores distributed in a spatial array, a cellular automata. The sum of the currents through such a set of pores models a noise analysis experiment. These currents were analyzed by using the optical correlation functions gn(tau) = (fn(t)fn(t + tau))/(f2(t))n, where f(t) are the current deviations around the mean current and () denotes the time average. These functions can be easily used to determine if the noise is Gaussian. If the noise is not Gaussian, they provide additional information not already contained in the power spectrum. When the pores do not interact with each other, the noise is Gaussian and the power spectrum a Lorentzian. When the pores interact in a strongly cooperative way the noise was still Gaussian and the power spectrum still a Lorentzian, but the usual analysis applied to such a case would over-estimate the single channel conductance. If the kinetics of the pore opening and closing vary on the time scale of the experiment then the relationship g2(tau) = 1 + 2[g1(tau)]2 is no longer satisfied.     

Production of active oxygen species (1O2 and O2-.) by psoralens and ultraviolet radiation (320-400 nm)

Furocoumarins (psoralens) are potent skin photosensitizing agents that are used in combination with long-wavelength ultraviolet radiation (320-400 nm) in the treatment of psoriasis and other skin diseases. Twelve linear and angular psoralens, capable of forming monofunctional and bifunctional adducts with DNA, were examined with a view to elucidate the role of 1O2 and O2-. in evoking skin photosensitization reactions and skin carcinogenesis. The results showed that both linear psoralens (capable of forming interstrand cross-links) and isopsoralens (angular, monofunctional type) and 3-carbethoxypsoralen (a linear and monofunctional type) produced 1O2 and O2-., although at varying degrees. Psoralen and 3-carbethoxypsoralen produced 1O2 greater than isopsoralens (angelicins). However, nonphotosensitizing angelicin, 5-methylangelicin, and 4,8-dimethyl-5'-carboxypsoralen produced 1O2 greater than 8-methoxypsoralen and 5-methoxypsoralen. The three monofunctional angelicin derivatives (isopsoralens) produced more O2-. than 8-methoxypsoralen, 5-methoxypsoralen, and 3,4'-dimethyl-8-methoxypsoralen. 3-Carbethoxypsoralen, a potent generator of 1O2 and a moderate producer of O2-., was highly photolabile. Until recently, skin photosensitization reactions (erythema, edema, damage to DNA or the membrane of cutaneous cells, the inhibition of scheduled DNA synthesis and skin carcinogenesis, etc.) were believed to involve photocyclo-addition of psoralens to DNA mediated by a type-I or anoxic reaction (a sensitizer-substrate interaction through the transfer of hydrogen atoms or electrons, but no direct involvement of molecular oxygen). Oxygen-dependent sensitized photodynamic reactions of type-II, involving the production of reactive oxygen (1O2 and O2-.), were believed not to mediate psoralen photosensitization reactions. We suggest that 1O2 and O2-. may also participate in skin photosensitization and cell membrane-damaging reactions. The fact that certain monofunctional isopsoralens produce 1O2 and O2-. at rates comparable to or better than bifunctional psoralens suggests that these reactive moieties of oxygen could play a major role in explaining their recently observed carcinogenic property and cell membrane-damaging reactions (e.g., edema or inflammation, etc.).     

Aggregation and inactivation of pancreatic cystatin by riboflavin-derived singlet oxygen and flavin triplet state: polyphenols as preventive agents

Caprine pancreatic thiol proteinase inhibitor (PTPI) a cystatin superfamily variant has high affinity for cysteine proteinases providing tight regulation of their proteolytic potential. Oxidative stress buildup in various pancreatic pathologies worsens the disease course often by disturbing the delicate balance between proteinases and their inhibitors. We aimed to study the effect of reactive oxygen species (ROS) on PTPI and to determine the potency of caffeic acid, curcumin and quercetin as agents against the inflicted damage. Fluorescence spectroscopy, polyacrylamide gel electrophoresis, and papain inhibitory assay revealed that photoilluminated riboflavin severely challenged the functional and structural integrity of the inhibitor. Three hundred and fifty micromolar affeic acid or quercetin prevented the damage. Curcumin, however, failed to reverse the changes completely. Conclusively, PTPI rendered dysfunctional by ROS may explain the increased necrotic damage to the host tissue. Also, dietary antioxidants can reverse the riboflavin-induced protein damage providing an economic and safe anti-ROS therapy.     

Correlation of the rates of singlet oxygen inactivation by phenols and their efficacy in the inhibition of neoplasms induced by benzo(a)pyrene

Prooxidant states, characterised by an increase in the intracellular concentration of activated forms of oxygen, are able to promote tumors. The inhibitory effects of synthetic phenolic compounds added to the diet of mice on benzo(a)pyrene-induced neoplasia of the forestomach have been determined by Wattenberg et al. The efficiency of this inhibition has been estimated quantitatively for each phenol, using the ratio R of the number of tumors per mouse in the treated group over the number of tumors per mouse in the control group. We have observed a linear correlation between the antitumoral efficiency (1-R) and the logarithm of the rate of quenching of singlet oxygen, k, y, by this family of phenols, log k being itself correlated with the half-wave oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced by benzo(a)pyrene, B(a)P. It should be pointed out that singlet oxygen can be generated via enzymatic reactions in the dark and thus might play a role in the formation of the ultimate carcinogenic metabolite B(a)P-7, 8-dihydroxy-9, 10-epoxide. The correlations described emphasize the interest in scaling the inhibitors of mutagenicity induced by polycyclic aromatic hydrocarbons with respect to their oxidation potentials. Our result is a first step towards a better understanding of the molecular reactions involved in chemically-induced neoplasia and in its prevention.     

Reactivities of diphenylfuran (a singlet oxygen trap) with singlet oxygen and hydroxyl radical in aqueous systems.

It has been studied whether 2,5-diphenylfuran is a specific singlet oxygen trap in aqueous systems. With certain ¹O₂ generating systems (Rose Bengal photooxygenation and NaOClH₂O₂ systems) and·OH generating systems (Fenton's reagent and acetaldehyde-xanthine oxidase system), diphenylfuran was chiefly converted in all cases to cis-dibenzoyl-ethylene, but not to trans-dibenzoylethylene. Low but detectable conversion of diphenylfuran to a hydroperoxide, probably a distinct ¹O₂-derived reaction in aqueous media, was found only in the Rose Bengal photooxygenation system.     

Optimization of the photodynamic inactivation of prions by a phthalocyanine photosensitizer: The crucial involvement of singlet oxygen

Prion disorders are fatal neurodegenerative diseases caused by the autocatalytic conversion of a natively occurring prion protein (PrP^C) into its misfolded infectious form (PrP^TSE). The proven resistance of PrP^TSE to common disinfection procedures increases the risk of prion transmission in medical settings. Herein, we present the effective photodynamic inactivation (PDI) of prions by disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO₃)₂) utilizing two custom‐built red light sources. The treatment eliminates PrP^TSE signal in infectious mouse brain homogenate with efficiency that depends on light intensity but has a low effect on the overall protein content. Importantly, singlet oxygen (O₂(¹Δ_g)) is the only species significantly photogenerated by AlPcOH(SO₃)₂, and it is responsible for the PDI of prions. More intensive light conditions show not only higher O₂(¹Δ_g) production but also decreases in AlPcOH(SO₃)₂ photostability. Our findings suggest that PDI by AlPcOH(SO₃)₂‐generated O₂(¹Δ_g) represents a promising approach for prion inactivation that may be useful in future decontamination strategies for delicate medical tools.