THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H₂O₂ is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o-Di and tri-hydroxy groups of polyphenols undergo oxidation by a free-radical mechanism and a green intermediate anion-radical with absorption Δ_max= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a-hydroxy-methylperoxide formed from H₂O₂ and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D₂O by a factor 6.5. Quenchers of O₂(¹Δ_g) and 1,3-di-phenylisobenzofurane diminish light intensity in non-aqueous solutions. The data suggest ¹O₂ participation in the observed chemiluminescence. Thermo-chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi-enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.     

CHEMICAL CHANGES LEADING TO CHEMILUMINESCENCE OF LUCIGENINE (DIMETHYLBIACRIDYLIUM NITRATE) AND OF SOME PHTHALAZINEDIONE DERIVATIVES

Abstract— Measurements of the redox potential of the chemiluminescent compound 10,10' dimethyl-9,9' biacridylium nitrate (-0.093 V) show that it is thermodynamically possible to reduce it with hydrogen peroxide or with ammonium hydroxide in alkaline solutions at equilibrium concentrations sufficiently high to account for the observed chemiluminescence. Reduction of the compound with ammonium hydroxide takes place much more slowly than the corresponding reaction with hydrogen peroxide so that when both redox couples (O₂/H₂O₂ and N₂H₄/NH₄OH) are present the hydrogen peroxide couple predominates if oxygen is supplied. It was shown that interference with the oxygen supply or its partial removal with nitrogen brings about an increase in chemiluminescence intensity in NH₄OH while increasing the concentration of oxygen diminished the intensity.
5-amino 2,3 phthalazine 1,4 dione (luminol) also appears to undergo a reduction following a two step oxidation. This is shown by the fact that when oxygen was supplied the chemiluminescence intensity was found to be directly proportional to the OH^- concentration while a typical titration curve with p K 11.7 is exhibited by the intensity when the oxygen supply is limited in mixtures of luminol and peroxydisulfate. The peroxide presumably arises in the first oxidation step. Amino peroxyphthalic anhydride is suggested as an intermediate which is reduced to the aminophthalate ion, the presumed emitter in the chemiluminescence.     

CHEMILUMINESCENCE IN THE PROCESS OF OXIDATIVE RING-OPENING OF PURPUROGALLINQUINONES

Abstract— The oxidation of purpurogalline (PPG) by alkaline solution of H₂O₂ pH 9–11 at 298°K is accompanied by chemiluminescence (CL) in the spectral range 400–600 nm with the maximum at 500 nm and quantum yield about 10^-6. The optimal concentrations of reactants with respect to maximal intensity are: 2 × 10^-4 M PPG, 10^-2 M NaOH, 1 M H₂O₂. Activation energy calculated from the maximum intensity of CL is 8.1×0.4 kcal/mole. Light emission occurs only when OH-groups of the phenolic ring of PPG undergo oxidation and the blue anion of o -PPG-quinone is formed. The rate that determines step in the reaction associated with luminescence is the nucleophilic attack of OOH^- ion on the blue anion of o -PPG-quinone. In this exergonic step (-ΔH = 63 to 230kcal/mole) the o - and/or p -quinone ring is opened and carbonyl derivatives of α-tropolone are produced. They display fluorescence in the region 400–600 nm. The fluorescence spectrum of the reaction mixture after oxidation of PPG is very close to that of CL. It is likely that carbonyl derivatives of α-tropolone are emitters of CL.     

CHEMILUMINESCENCE FROM AUTOXIDIZING 6-HYDROXYDOPAMINE: THE INVOLVEMENT OF ACTIVATED FORMS OF OXYGEN

Abstract— The autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (20 μ M ) gave rise to a chemiluminescence which was greatly stimulated by FeSO₄ (20 μ M ) or by hydrogen peroxide addition (20 μ M to 2 m M ). The luminescence of both 6-hydroxydopamine alone or 6-hydroxydopamine plus hydrogen peroxide was strongly inhibited by catalase and by superoxide dismutase (both at 10 μg/m/); bovine serum albumin at 10 μg/m/ had no inhibitory effect. The luminescence was also strongly inhibited by several potent hydroxyl radical trapping agents and also by low concentrations of the ¹O₂ quencher DABCO (l,4-diazabicyclo-2.2.2.-octane). Chemiluminescence was greatly enhanced in D₂O, a solvent in which ¹O₂ has a prolonged lifetime. These data demonstrate the involvement of hydrogen peroxide, the superoxide radical and the hydroxyl radical in the chemiluminescence. The data are also consistent with some role for ¹O₂.     

CHEMILUMINESCENCE OF SOME REACTIONS WITH MOLECULAR OXYGEN

Abstract— Many inorganic oxidation reactions involving a variety of oxidizing agents show chemiluminescence in the spectral region of 400–600 mp. Excited O₂O₂-associates are assumed to be the emitting molecules formed by bimolecular recombination of HO _x O₂-radicals. Oxidation of sodium sulfite with molecular oxygen in the presence of Cu- and Fe-basic oxides shows chemiluminescence which originates from the reactions of the heavy metal complexes of OH- and O, H-radicals with O₂. The simplest way of producing a radical from O₂ is by the uptake of one electron. Suitable electron donors, such as hydro-quinones and semiquinones, emit light if treated with oxygen. In certain organic solvents OH- can also act as electron donor. Its presence causes the formation of semiquinones from quinones in the absence of oxygen. The chemiluminescence which is observable upon treatment of alkaline dimethylsulfoxide, dioxane, pyridine-water and alcohol-water mixtures with oxygen is also attributable to electron transfer from OH- to O₂.     

CHEMILUMINESCENCE AND FLUORESCENCE OF THE EUROPIUM IONS-ADENINE NUCLEOTIDES SYSTEM AND ITS POSSIBLE BIOLOGICAL SIGNIFICANCE

Abstract— Chemiluminescence of the Eu(II)/Eu(III)-adenine nucleotide-H₂O₂ system and fluorescence of the Eu(III)-adenosine triphosphate system have been investigated. The spectral distribution of the chemiluminescence emission has shown an occurrence of three main bands (Λ=470–480,590–620 and ca. 700 nm). The energy transfer process from the adenosine triphosphate molecules to the Eu(III) ions has been observed in the fluorescence spectrum. The examined chemiluminescence and fluorescence spectra show that these both kinds of emission originate from the ⁵ D _***τ⁷F_*** ( n =1–4) transitions in the Eu(III) ions.     

VISIBLE CHEMILUMINESCENCE ASSOCIATED WITH THE REACTION BETWEEN METHEMOGLOBIN OR OXYHEMOGLOBIN WITH HYDROGEN PEROXIDE

Abstract Visible chemiluminescence is emitted in the irreversible deactivation of hemoglobin or methemoglobin with excess H₂O₂. The emission takes place in two phases. The most intense one lasts a few seconds and is followed by a second phase of lower intensity that remains for longer periods. This second phase presents chaotic or sustained oscillations. Free radicals are implicated in the luminescent process since the emission can be reduced by free radical scavengers such as 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox) or ascorbic acid. These additives lead to a delay in reaching the maximum intensity, which can be related to their consumption, implying substantial recycling of the hemoprotein. Chemiluminescence is also observed in the oxidation of hemin by H₂O₂, suggesting a role for the heme group in the processes leading to the excited state production. The lower intensity observed in the presence of hemin can be related to the contribution of the globin chains.     

SPECTRAL CHARACTERISTICS AND MECHANISM OF CHEMILUMINESCENCE FROM TRYPTOPHAN SOLUTIONS INDUCED BY UV-IRRADIATION

The spectral distribution of the chemiluminescence, fluorescence and phosphorescence of tryptophan aqueous solutions irradiated with high and low pressure mercury lamps has been measured. The blue emission bands in the region of 380–520 nm observed both in the chemi- and photoluminescence, as well as an absorbance increase at 230 and 330 nm, indicate oxidative degradation of tryptophan leading to the formation of derivatives of N-formylkynurenine, xanthurenic and anthranilic acids. Red emission bands at 630 and 705 nm in the spectrum of the chemiluminescence, an enhancement of light intensity by D₂O and its decrease by NaN₃ and DABCO suggest a partial contribution of O₂(¹Δ_g) to the photooxidation and chemiluminescence of tryptophan. The enthalpy of the exergonic reactions, leading to the formation of luminescing products, was calculated to average -270 kJ-mol.     

CHEMILUMINESCENCE INDUCED BY OXIDATION OF TRYPTOPHAN BY SINGLET OXYGEN AND BY HYPOCHLOROUS ACID. IMPLICATIONS IN THE LUMINESCENCE EMITTED IN PHAGOCYTOSIS

Abstract— The mechanism of the chemiluminescence emitted during phagocytosis of opsonized zymosan was investigated. The use of an artificial system generating singlet oxygen permitted the distinction between the respective roles of O₂ and HOCl in the light production via the reactions with opsonized zymosan, amino-acids and peptides. The reaction of free hypochlorous acid, produced by the myeloperoxidase H₂ O₂-Cl- system, with free tryptophan appears to be a likely path for the production of chemiluminescence. Implications for the mechanism of in vivo chemiluminescence during phagocytosis are considered.     

CHEMILUMINESCENCE IN THE OXIDATION OF 6-HYDROXYDOPAMINE: EFFECTS OF LUCIGENIN, SCAVENGERS OF ACTIVE OXYGEN, METAL CHELATORS AND THE PRESENCE OF OXYGEN

Abstract— Maximum chemiluminescence in a system containing 6-hydroxydopamine (6-OHDA) and H₂O₂ required the addition of Fe²⁺:EDTA, oxygen, and lucigenin. In this system luminescence was strongly inhibited by catalase (91% inhibition) or 50 m M mannitol (83%), whereas superoxide dismutase or ascorbate did not significantly change the reaction rate. In the absence of lucigenin, 50 m M mannitol (78%), catalase (76%), or ascorbate (73%) inhibited strongly, while superoxide dismutase inhibited by 60%. Removing EDTA from the lucigenin-containing system caused a 79% decrease in luminescence, while the substitution of desferoxamine for EDTA decreased luminescence by 55%. In the presence of desferoxamine plus EDTA the luminescence increased by 30% in comparison with that seen with EDTA alone. Luminescence in the system containing 6-hydroxydopamine, H₂O₂, Fe²⁺:EDTA and lucigenin required the presence of oxygen (93% inhibition anaerobically), consistent with a mechanism involving reductive oxygenation of the lucigenin. It is concluded that luminescence in the presence of lucigenin involves a substantial contribution from H₂O₂ and Fe²⁺ mediated by a mannitol-sensitive intermediate (conceivably Fenton-derived hydroxyl radicals). In the absence of lucigenin, superoxide and an ascorbate-labile component are additional important participants in the process.     

QUENCHING OF PEROXIDIZED LUMINOL CHEMILUMINESCENCE BY REDUCED PYRIDINE NUCLEOTIDES

Abstract— Reduced pyridine nucleotides were observed to cause a delay as well as a diminution of light emission from peroxidized luminol at pH 6.5. Other reductants were found to have similar effects. Neither superoxide nor hydroxyl radical scavengers quenched chemiluminescence of luminol in the presence of horseradish peroxidase and H₂O₂. A scheme in which reductants such as NADH and NADPH prevent peroxidase from oxidizing luminol to aminophthalate is proposed. Moreover, it is concluded that neither O₂⁻nor OH' play a role in the peroxidation of luminol by horseradish peroxidase.     

PHOTOLYSIS OF AQUEOUS SOLUTIONS OF POTASSIUM CIS-DIAQUOBIS (OXALATO) CHROMATE (III)

Abstract— The photolysis of aqueous solutions of cis -[Cr(C₂O₄)₂(H₂O)₂]- at 254 nm and pH 4 produced CO₂ and H₂ in nearly equal yields. The quantum yield of hydrogen, φ², increased by 9% and the yield of carbon dioxide, φ, by 65% as the pH was lowered from 4 to I. The total gas yield, φ_gas, decreased in the presence of added oxalate or chromium (II) ions and when the light intensity was lowered. The gas yield in D₂O was appreciably higher than in H₂O. The variation of φ_gas with pH (D) and with added oxalate ion was roughly parallel in the two liquid media. The gas yield increased in the series:
A tentative mechanism is suggested to explain the results.     

INHIBITION OF ETHYL BENZENE OXIDATION BY STABLE IMINOXY RADICALS, STUDIED BY CHEMILUMINESCENCE

Abstract— The inhibition by stable radicals having the structure (X-O)₂NO of the initiated oxidation of ethyl benzene was studied. According to a formal kinetic scheme, verified by both theoretical computer calculations and an experimental chemiluminescence method, the rate constants of the interaction of peroxyradicals with iminoxy radicals were estimated at 60°C to be: 2.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₃ and 3.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₂CH₃.     

BIOLOGICAL CHEMILUMINESCENCE

Abstract
The nucleobase 5-methylcytosine ( I ) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine ( II ), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine ( Ib ) and 3-(2- erythro - d -pentopyranos-1-yl) amino-2-methylacrylamidine ( IIb ) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (¹H and ¹³C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib , confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2–¹³C]cytosine, hydrolysis of N -carbomethoxy-3-amino-2-methylacryl-amidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I ). The quantum yield for formation of Ib was determined to be 1.8 × 10^-3at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 × 10^-3 at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 × 10^-3 at pH 7.0 ( Ib ) and 0.6 × 10^-3 at pH 5.0 ( IIb ). The mechanism of formation of Ib (or IIb ) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II ), followed by ring opening and decarboxylation of an intermediate carbamic acid.     

FORMATION AND CHEMILUMINESCENT DECOMPOSITION OF DIOXETANES IN THE GAS PHASE

Abstract— High resolution chemiluminescence spectra have been obtained of the singlet electronically excited products of O₂(¹Δ) plus alkene, dioxetane forming, reactions. The experiments were conducted in a flow apparatus at pressures of 1–5 torr. The spectra are a measure of the unrelaxed initial distribution of energy in the excited product. Results are reported for ethylene, 1, 1-difluoroethylene. methyl vinyl ether, ethyl vinyl ether, n -butyl vinyl ether, ketene, ketene-d₂, allene, unsymdimethyl allene, dimethyl ketene, 2-methoxy propene, 1-ethoxy propene, 2-bromo propene, and N, N- dimethyl isobutenyl amine. Chemiluminescence activation energies, representing the cycloaddition process, and absolute quantum yields for singlet excited product, ranging from 10^--⁴ to 2.5 × 10^--². are reported for 10 alkenes. Several of the reactions, 1,1-difluoroethylene, ketene, ethylene and allene give formaldehyde ¹ nπ* product with excess vibrational-rotational energy and a higher quantum yield than reactions not displaying this phenomenon. This is an indication of at least partially statistical partitioning of the energy in excess of that needed to electronically excite the formaldehyde. The experiments with ketene and ketene-d₂ provide the first evidence for the existence of unsubstituted 1,2-dioxetanone. The results from several of the experiments, particularly those with 2-methoxy propene and I-ethoxy propene are consistent with the mechanism of Goddard, which predicts regioselective and stereoselective attack of O₂(¹Δ) upon alkoxy substituted alkenes having allylic hydrogen.     

QUANTUM YIELD OF SINGLET OXYGEN PRODUCTION BY XANTHENE DERIVATIVES

The singlet oxygen quantum yield (φ₁o₂) of 11 purified fluorescein derivatives was determined by reaction with singlet oxygen acceptors in aqueous and ethanolic solutions; in both solvents φ₁o₂ was enhanced with increasing halogenation. Tryptophan and 2,2,6,6-tetramethylpiperidone were found to be unadapted for the determination of φ₁o₂, in our systems; however, the use of 9.10-dipropionic anthracene acid andp-nitrosodimethylaniline in conjunction with imidazole derivatives was suitable for ¹O₂ detection in water. Both methods lead to results in excellent agreement. As in ethanol. φ₁o₂, was equal to the triplet state quantum yield (φ_T), the comparison between the two solvents showed that φ_T in water was greater than in ethanol. The comparison between our values obtained with polychromatic light with published data obtained with monochromatic light suggests that the triplet quantum yield of fluorescein derivatives is wavelength independent.     

ACTIVATION OF LUCIGENIN CHEMILUMINESCENCE BY SERRATIA MARCESCENS

Abstract Cell–free extracts and intact cells of Serratia marcescens were found to activate lucigenin (10,10 -dimethy1-9,9-biacridylium nitrate) chemiluminescence in the absence of either added H²O² or alkali. Light emission proceeded in alcoholic solvents and, in general, the intensity decreased with increasing length of the alcoholic carbon chain. Th e intensity of bacterially activated lucigenin chemiluminescence increased in a logarithmic linear manner with increasing methanol concentrations, maximum intensities occurring with 90% methanol. Other organic lucigenin solvents also supported the bacterially activated light emission process, although not to the same extent as 90% methanol. The addition of KOH to methanol failed to enhance chemiluminescence. The luminescent process was charaterized by the attainment of peak light emission three seconds after the initiation of the reaction, followed by rapid decay to a low constant light level. The bacterial activation of lucigening chemiluminescence was found to be enhanced by the inclusion by the inclusion of fluorescein in the neutral methanol solvent.     

QUANTIFICATION OF THE SOLVENT EFFECTS ON THE TRIPLET QUANTUM YIELD OF PSORALEN BY THE "LINEAR SOLVATION ENERGY RELATIONSHIP"

Triplet formation quantum yields (Φ_τ) of psoralen in a set of 17 pure solvents ranging from n -hexane to water and in dioxane: water mixtures were obtained by nanosecond laser flash photolysis. The triplet yield increases with solvent polarity. The extremum values are 0.009 and 0.545 in n -hexane and water, respectively. Good correlations of the experimental Φ_τ values with empirical "polarity" scales (Dimroth/Reichardt's E_T [30], Kamlet/Taft's solva-tochromic parameters β, and α, and Swains acity/basity A_S/B_S) were obtained: Ln(φ_T^-1 - 1) = 8.86 - 0.143E_T(30) Ln(φ_T^-1 - 1) = 4.40 - 2.34_τ - 1.70α Ln(φ_T^-1 - 1) = 4.65 - 3.72A_s - 1.12B_s The results are discussed in terms of the sensitivity of psoralen triplet quantum yield to solvent polarity and hydrogen-bonding abilities.