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.     

RADIATION ACTIVATION OF CARCINOGENS AND THE ROLE OF OH AND O2-

Abstract— Radiation-induced covalent binding of labelled carcinogens to DNA has been investigated under a variety of conditions using ultrafiltration or millipore filtration of TCA precipitable complexes. High yields of carcinogen binding at high DNA concentrations are also observed for a variety of small molecules and are not carcinogen-specific. At high carcinogen concentrations, radiation-induced unstable electrophilic carcinogenic species are produced, and undergo free-radical reactions which simulate cellular redox reactions involved in metabolic carcinogen activation, leading to the formation of covalently bound carcinogen adducts to DNA as a potential target macromolecule. The yields of carcinogen-DNA adducts increase linearly with dose and depend upon carcinogen concentration. The results of scavenger studies indicate that the oxidising species O₂^- and OH are the principal activating species. Rate constants for the selective radiation-induced oxidation reactions of various chemical carcinogens with superoxide have been measured by a competition kinetic method using pulse radiolysis. The relatively long-lived superoxide radical reacts with carcinogens at a rate which is two orders of magnitude slower than the diffusion-controlled rate for the hydroxyl radical, thus allowing a measure of O₂^- specificity in the presence of competing reactants within the cell.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

THE STUDY OF LUMINOL-H_2O_2-IO_4~--S~(2-)CHEMILUMINESCENCE SYSTEM AND ITS APPLICATION

This paper reports a new chemiluminescence system i.e.luminol-H_2O_2-IO_4~- catalyzed by sulphide ion(S~(2-)).Based on this catalysis,a newchemiluminescence(CL)method for the determination of trace S~(2-)is pro-posed.The detection limit is 0.2μg/LS~(2-),the linear dynamic range is 5μgto 100βμg/L S~(2-)and the variation coefficient at an sulphide concentrationof 100μg/L S~(2-)is 4.5%(n=10).The method has been satisfactory applied tothe determination of trace sulphide ion in water.     

PHOTOCHEMICALLY INDUCED BINDING OF Rh(phen)2Cl2+ TO DNA†

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

SEARCH FOR SINGLET OXYGEN LUMINESCENCE IN THE DISPROPORTIONATION OF HO2/O2

Abstract— During the reaction HO₂+ HO₂ (or O₂^-) = H₂O₂+ O₂ in aqueous solution, no luminescence in the region 620–720 nm, expected if the product O₂ were formed in a singlet state, could be detected. If any singlet O₂ is formed, its yield must be less than 10%. Faint luminescence, sometimes found at shorter wavelengths, was shown to arise from reaction of HO₂ with impurities in the reagents present.     

A MORE EXACT THEORETICAL INTERPRETATION OF RECENT EXPERIMENTAL DATA OBTAINED USING THE SEPARATED SENSITIZER AND SUBSTRATE METHOD TO INVESTIGATE O2(lδg) INTERACTIONS. POSSIBLE IMPORTANCE OF O2(1σg+)

Abstract— Recent experimental data obtained using the separated sensitizer and substrate method to investigate the interaction of O₂(¹δ_g) with various substances has been re-interpreted by means of a more complete theory. Comparison of experimental and recalculated values of the dependence of relative reaction rates on the sensitizer-substrate separation indicate general accord for experiments in which singlet oxygen acceptors in aqueous solution were used. The presumption is therefore that singlet molecular oxygen O₂(¹δ_g) is indeed the active oxidizing agent and that the theory presented and experiment are entirely in agreement.
For experiments in which bacterial targets were used a very distinct disagreement between theory and experiment is evident, the conclusion being that the kill rate does not depend linearly on the O₂(¹δ_g) concentration in the immediate proximity of the bacteria. However, the data is consistent with a quadratic dependence on the ¹δ_g concentration. A possible conclusion therefore is that the cytotoxic species is actually O₂(¹σ⁺_g), formed by an energy pooling reaction involving two O₂(¹δ_g) molecules.     

THERMODYNAMIC FUNCTIONS FOR SINGLET MOLECULAR OXYGEN, O2(1△g) and O2 (1σ8+)

Abstract— A correction is offered to the approximate values previously given by Mendenhall (1978) for the enthalpy of formation and entropy of O₂(a₁Δ_g) and O₂(b₁₊) between 298 and 1500 K. Accurate values have been calculated for the functions together with the equilibrium constants for the formation of these species from O₂(X₃σ_g-).     

Na+/K+ TRANSPORT AND PHOTOSENSITIVITY OF THE COLORLESS FLAGELLATE Peranema trichophorum (Euglenida)

Abstract— Perimycin, ouabain and elevation of extracellular K⁺ concentrations cause an increase in the fluence rate thresholds (white light) for the step-up photophobic response in Peranema trichophorum . Elevation of extracellular Na⁺ concentration decreases the thresholds for this response in comparison to the control level. The fluence rate threshold of perimycin-treated cells increases before the side effect of an antibiotic action appears. Removal of K⁺ ions from the medium of K⁺-treated cells to a concentration of 1 mM depresses the threshold for the step-up response to the control level. By addition of K⁺ or Na ⁺ ions to perimycin- or ouabain-treated cells the threshold returns to the control value. It is suggested that the flagellar and cell membrane are responsible for changes of P. trichophorum photosensitivity.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.     

SIMPLE EXPLANATION OF THE MISSES IN THE COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION

Abstract —In the model of Forbush et at. (1971) the observed damping of the flash yield sequence of photosynthetic O₂ evolution was related to a certain percentage of ‘misses’ (α; i.e. centers not converted). The possibility of a miss was supposed to be equal for all states S_0.1,2,3. We propose a new model and a new recurrence law that gives better quantitative agreement with the O₂ yield oscillations observed in Chlorella during a sequence of flashes. We find a better fit with all experimental results by assuming very unequal misses; the misses occur nearly exclusively on S₂ (and also sometimes on S₃). In the simpler case of only one miss on one state, half of S₂ exists as an inactive form S₂₊^- because it is in apparent equilibrium with pool A. The active form of S₂ is converted to S₃ in a flash and the unchanged inactive form S₂₊^- explains the miss: S ₁ hv→S₂₊^-=S₂hv→S₃ (S₂₊^- is a transition state between S₁ to S₂ associated with Q^-). In the dark, the apparent equilibrium constant K_A between pool A and Q (i.e. S₀, S₁ in the dark) is very large; this explains why there is no miss on these states. In light, the experimental value of K_A between pool A and Q (i.e. S₂, S₃ in the light) is 1, and this explains why the misses are large for states S₂, S₃; i.e., S₂₊^-/S2- 1 and sometimes S₃₊^-/S₃?0–1. This new model predicts that the total number of active states ΣS_i=S₀+S₁+S₂+S₃ is an oscillating function of the flash number. This sum 2S, is also the number of trapping centers for excitons. As fluorescence is proportional to excitons that are not trapped, our model explains why the fluorescence oscillates as a function of the flash number. We find also that the initial rates of O₂ evolution after (n - 1) flashes vs the 02 yield of the n^th flash are not exactly on a straight line, which also favors our model.     

EPR and Spectrophotometric Studies of Free Radicals (O2°−, °OH, BPD-MA°−) and Singlet Oxygen (1O2) Generated by Irradiation of Benzoporphyrin Derivative Monoacid Ring A

Abstract— Benzoporphyrin derivative monoacid ring A (BPD-MA), a chlorin-type molecule, is a new photosensitizer currently in phase II clinical trials for the treatment by pho-todynamic therapy of cancerous lesions, psoriasis and pathologic neovascularization. The photochemistry (type I and/or II) of BPD-MA has been studied in homogeneous solution and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) using electron paramagnetic resonance and spectrophotometric methods. When oxygen-saturated solutions of BPD-MA were illuminated with 690 nm light, singlet oxygen (¹O₂), superoxide anion radical (O₂^?), hydroxyl radical (OH) and hydrogen peroxide (H₂O₂) were formed. The BPD-MA generates ¹O₂ with a quantum yield of ca 0.81 in ethanolic solution. The quantum yield does not change upon incorporation of BPD-MA into liposomes of DPPC. The superoxide anion radical was generated by the BPD-MA anion radical (BPD-MA^?) via electron transfer to oxygen, and this process was significantly enhanced by the presence of electron donors. The rate of production of 0₂ was also dependent on the concentration of BPD-MA used (3-100 μM). The quantum yield of O₂^?was found to be 0.011 and 0.025 in aqueous solution and DPPC liposomes, respectively. Moreover, O₂^_upon dis-proportionation can generate H₂O₂ and ultimately the highly reactive OH via the Fenton reaction. In anaerobic homogeneous solution, BPD-MA^?was predominantly photoproduced via the self-electron transfer between the excited- and ground-state species. The presence of an electron donor significantly promotes the reduced form of BPD-MA. These findings suggest that the photodynamic action of BPD-MA may proceed via both type I and type II mechanisms.     

PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2

The photophysics of the complex forming reaction between Quin-2 and Ca²⁺ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca²⁺ buffer: k₀₁= 8.6 times 10⁸ s^?1, k₂₁= 1 times 10¹¹M^?1 s^?1, k₀₂= 8.8 times 10⁷ s^?1, k₁₂= 4 times 10⁴ s^?1. k₀₁ and k₀₂ denote the respective deactivation rate constants of the Ca²⁺ free and bound forms of Quin-2 in the excited state. The constant k₂₁ represents the second-order rate constant of binding of Ca²⁺ and Quin-2 in the excited state while k₁₂ is the first-order rate constant of dissociation of the excited Ca²⁺:Quin-2 complex. From the estimated values of k₁₂ and k₂₁ the dissociation constant K_d* in the excited state was calculated. It was found that pK_d* (6.4) is slightly smaller than pK_d (7.2). There was no interference of the excited-state complex forming reaction with the determination of K_d. Intracellular Ca²⁺ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca²⁺ indicator.     

Comparative Study of Ru(bpz)32+Ru(bipy)32+and Ru(bpz)2CI2 as Photosensitizers of DNA Cleavage and Adduct Formation

Abstract— The efficiency of ruthenium complexes for photosensitizing DNA damage depends on the oxidizing character of their ligands. Here we report on the difference in behavior of tris(2.2'-bipyrazyl)ruthenium(II) (Ru[bpz]₃²+), tris(2,2′-bipyridyl)ruthenium(II) (Ru[bipy]₃²+) and cis-dichlorobis(2,2′-bipyrazyl)ruthenium(II) (Ru[bpz]₂Cl₂). Upon irradiation at 436 nm, Ru(bpz)₃²+was far less stable than Ru(bipy)₃²+. Ru(bpz)₃²+in phosphate buffer containing NaCl undergoes a photoanation reaction leading to the formation of Ru(bpz)₂Cl₂, as previously reported also in organic media. In the presence of phage φX174 DNA, Ru(bpz)₃²+photosensitized the formation of single strand breaks with an efficiency that was, at the beginning of irradiation, similar to that of Ru(bipy)₃²+. After 8 min of irradiation, the cleavage efficiency of Ru(bpz)₃²+reached a plateau that may correspond to its photode-composition. For the same conditions, Ru(bpz)₂Cl₂ did not induce DNA breakage. Scavenging experiments showed that, in the presence of oxygen, DNA cleavage induced by Ru(bpz)₃²+partly resulted from the formation of singlet oxygen and hydroxyl radical while in the absence of oxygen an additionnal mechanism involving electron transfer between the excited state of the ruthenium complex and DNA is proposed. The ICP measurement showed that Ru(bpz)₃²+and Ru(bpz)₂Cl₂ gave rise to covalent binding onto DNA in contrast with Ru(bipy)₃²+, which did not bind to DNA under the experimental conditions. The results are discussed with regard to the potential use of these photosensitizers in phototherapy.     

DETERMINATION OF THE BINDING CONSTANT OF H14 CO−3 TO THE PHOTOSYSTEM II COMPLEX IN MAIZE CHLOROPLASTS: EFFECTS OF INHIBITORS AND LIGHT

The binding (dissociation) constant for HCO^?₃ to the photosystem II complex in maize chloroplasts is approximately 80 μM. One HCO^?₃ binds per 500–600 chlorophyll molecules. In the dark, formate is a competitive inhibitor of HCO^?₃ binding, while 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea (DCMU) inhibits HCO^?₃ binding non-competitively. Light decreases HCO^?₃ binding in the presence of formate. Light increases the binding of HCO^?₃ in the presence of DCMU. The high binding constant for HCO, discriminates strongly among the various hypotheses attempting to explain the “bicarbonate-effect” on photosystem II. The proposal by Stemler and Jursinic (Arch. Biochem. Biophys. 221, 227–237 1983), that HCO^?₃ is one of a class of monovalent anionic inhibitors of photosystem II, is favored. These anions compete for a specific binding site on the photosystem II complex.     

PROTON, Mg2+ AND PROTEIN AS COMPETING LIGANDS FOR THE FLUORESCENT PROBE, MAG-INDO-1: A FIRST STEP TO THE QUANTIFICATION OF INTRACELLULAR Mg2+ CONCENTRATION

Abstract Increasing evidence of the role of magnesium in various cellular mechanisms has led to the need to develop an accurate method for the evaluation of magnesium concentration in cells. 1H-indole-6-carboxylic acid, 2–(4- bis- [carboxymethyl]amino-3–[carboxy]ethoxy) (mag-indo-1) is used as a fluorescent indicator for ionized magnesium concentration. A physicochemical study of this probe has pointed out (1) that at concentrations higher than 10 μ M , the presence of dimers can alter the different equilibria and (2) at concentrations, avoiding the dimer (≤ 10 μ M ), three fluorescent forms are in equilibrium with the deprotonated form of mag-indo-1 (L), which are the protonated form LH, the magnesium-bound form LM and the protein-bound form LP. A model is proposed that takes into account the equilibria between the four species. In a solution containing magnesium and protein, a complex fluorescence spectrum can be resolved by a combination of the three fluorescence spectra (L, LM, LP). However, under these conditions, the LH fluorescence spectrum is not taken into account for the spectral resolution. Finally, from the contribution of characteristic fluorescence spectra in the experimental fluorescence spectrum, the magnesium concentration can be estimated with accuracy. Such a method should be further applied to magnesium determination in different cell lines.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES

Abstract
In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M N and N bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.