BIOLUMINESCENCE FROM THE REACTION OF FMN, H2O2 AND LONG CHAIN ALDEHYDE WITH BACTERIAL LUCIFERASE

Abstract— The bioluminescent oxidation of reduced flavin mononucleotide by bacterial luciferase involves a long-lived flavoenzyme intermediate whose chromophore has been postulated to be the 4a-sub-stituted peroxy anion of reduced flavin. Reaction of long chain aldehyde with this intermediate results in light emission and formation of the corresponding acid. These experiments show that the typical aldehyde-dependent, luciferase-catalyzed bioluminescence can also be obtained starting with FMN and H₂O₂ instead of FMNH₂ and O₂. We postulate that the 4a-peroxy anion intermediate is formed directly by attack of H₂O₂ on FMN. The latter may be bound to luciferase. An enzyme bound intermediate is formed which by kinetic analysis, flavin specificity for luminescence, aldehyde dependence, and bioluminescent emission spectrum appears to be identical with the species generated by reaction of FMNH, and O₂ with luciferase. The quantum yield of the H₂O₂-_- and FMN-initiated biolumlnescence is low but can be enhanced by certain metal ions, which also stimulate a chemiluminescent reaction of oxidized flavin with H₂O₂. The peak of this chemiluminescence. however, appears to be at a shorter wavelength than that (490 nm) of the bioluminescence.     

Chemiluminescence of Horseradish Peroxidase and Acetaldehyde Related with Gallic Acid and Hydrogen Peroxide Interaction

Abstract— This study focuses on the fact that the chemiluminescence in the visible region is emitted from the H₂O₂/gallic acid/ horseradish peroxidase (HRP) and the H₂O₂/gallic acid acetaldehyde (MeCHO) systems. The concentration dependence of chemiluminescence intensity that led to the different response of HRP and MeCHO toward H₂O₂ indicates that the photon emission participates with peroxidase activity including an electron transfer reaction. From our experimental results, in this study, we postulated a reaction process for chemiluminescence based on a one-electron redox shuttle from H₂O₂ by peroxidase. The photon intensity and spectra data from the H₂O₂/ HRP and the H₂O₂/MeCHO systems with various cate-chins were not only affected by HRP and MeCHO but also corresponded with the chemical structure of cate-chins. The energy calculated from the spectra is 47–64 kcal/mol. These results suggested that the chemiluminescence of both systems arose from excited carbonyl compounds produced by an intermediate of the alkyl radical and the metal-bound hydroxyl (compound II species). Hydroxyl radical inhibition, showing a notable increase from the gallic acid addition, makes the decay of the hydroxyl form of heme iron the most likely candidate for the chemiluminescence.     

CATALYTIC ACTION AND DESTRUCTION OF PROTOHEMATIN DURING PEROXIDE DEPENDENT LUMINOL CHEMILUMINESCENCE

Abstract— The catalytic action of protohematin was studied during the H₂O₂-dependent chemiluminescent luminol reaction. In spite of the fact that the catalyst was ultimately inactivated, the average protohematin molecule catalyzed the consumption of about 10³ molecules of luminol. The inactivation of catalyst and the initial consumption of luminol were studied during the luminescent reaction with different concentrations of reactants. A scheme accounting for the experimental observations is proposed. The formation of a primary protohematin-H₂O₂ complex is followed by binding of luminol, resulting in a ternary complex. A nucleophilic attack by a second molecule of H₂O₂ on the luminol molecule results in light emission from excited aminophthalate via a hypothetical peroxide adduct. The destruction of protohematin occurs via the attack of H₂O₂ on the porphyrin structure of the protohematin-H₂O₂ complex. Second order rate constants for the destruction of protohematin, the formation of the luminol complex and the nucleophilic attack of H₂O₂ are presented.     

Near-infrared Light Emission from Nanomolar-level Singlet Molecular Oxygen Generated with an Ultra low Concentration Mixture of Hypochlorite and Hydrogen Peroxide

Abstract— We report the detection of a weak near-infrared light emission originating from 8 nM singlet molecular oxygen (¹O₂) produced in a mixture of 1 m M hypochlorite (OC1^-) and 8 n M hydrogen peroxide (H₂O₂). The measurements were made with a highly sensitive detection system for ultraweak light emission in the 1.0-1.5 μm wavelength region. The emission intensity exhibited linear dependence for H₂O₂ concentrations in the range of 8-670 n M . The mixture containing a lower concentration (33 μ M ) of OCl^- pseudocontinuously emitted near-infrared light for 5 s. The rate constant for ¹O₂ production obtained from the kinetic analysis agrees with that previously reported. Our results demonstrate the possibility of measuring very low concentrations of ¹O₂ in a OCi-/H₂O² mixture as well as ¹O₂ production in intact living systems.     

INACTIVATION OF PHAGE BY NEAR-ULTRAVIOLET RADIATION AND HYDROGEN PEROXIDE

A series of phage with different genomes (both single-stranded and double-stranded RNA and DNA) was inactivated with hydrogen peroxide (H₂O₂) in various combinations with far-ultraviolet (FUV) and near-ultraviolet (NUV) radiations. In every case but one (a lipid-coated phage), a sublethal H₂O₂ concentration greatly enhanced killing by NUV but not FUV. Moreover, this NUV/H₂O₂ synergism was oxygen independent and there was little if any host cell reactivation upon NUV plus H₂O₂ inactivation. These results suggest that these phage are inactivated by a common mechanism irrespective of nucleic acid composition, but that some phage genomes may be more vulnerable to NUV/H₂O₂ inactivation than others.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

Development of Refractoriness of Induced Human Heme Oxygenase-1 Gene Expression to Reinduction by UVA Irradiation and Hemin

Abstract— Using primary human fibroblasts we have observed the existence of an acquired refractoriness of the heme oxygenase-1 gene to induction by a second dose of UVA (320-380 nm) radiation. We studied the kinetics of development of refractoriness over a time interval of up to 72 h between the first inducing event and the second (challenge) dose. Complete refractoriness was observed at 48 h. We also studied development of refractoriness after UVA, sodium arsenite and H₂O₂ treatment in all possible combinations and demonstrated that only UVA led to refractoriness. Ultraviolet radiation induced partial refractoriness to H₂O₂ induction but did not change the response to sodium arsenite. In an investigation of the mechanism of development of refractoriness we used the heme oxygenase inhibitor, tin-protoporphyrin IX and showed that induction of heme oxygenase enzymatic activity is a crucial step. However, the induction of ferritin, which is known to play a key role in protection against oxidative stress, did not appear to be involved. Damage to membranes is also probably not involved in the refractoriness mechanism. Because either hemin alone or UVA radiation are able to lead to a refractoriness of the heme oxygenase-1 gene to reinduction by a second exposure to one or the other agent in human fibroblasts, we conclude that heme, or an as yet unidentified heme derivative, is involved in the refractoriness response.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

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.     

PHOTODYNAMIC GENERATION OF HYDROXYL RADICALS BY HEMATOPORPHYRIN DERIVATIVE AND LIGHT

In a reaction mixture containing hematoporphyrin derivative, deoxyribose, Fe³⁺-EDTA and either methionine or tryptophan, hydroxyl radicals were formed during illumination with visible light. When either hematoporphyrin derivative, Fe³⁺-EDTA or the amino acid was omitted from the reaction mixture, the generation of hydroxyl radicals ceased. These observations suggest an iron-catalyzed Haber-Weiss reaction, involving superoxide and hydrogen peroxide in the generation of hydroxyl radicals. It could be shown that with methionine H₂O₂ was indeed an essential intermediate in the reaction sequence. With tryptophan, however, H₂O₂, was not generated. Apparently a photooxidation product of tryptophan could replace H₂O₂ in the OH-generating reaction with Fe²⁺-EDTA. Although superoxide was generated in the reaction mixture, it was not an indispensable intermediate. Apparently a porphyrin radical, formed via photoexcitation of hematoporphyrin derivative, could replace superoxide in the Haber-Weiss reaction.     

Sister chromatid exchanges and chromosome aberrations in human cells induced by H2O2 and other photoproducts generated in fluorescent light-exposed medium

Exposure of Dulbecco's modified Eagle's tissue culture medium to visible fluorescent light generated photoproducts toxic to human cells in culture. Toxicity manifested at the chromosome level was increased chromosome aberrations and sister chromatid exchanges in cells exposed to the photoproducts. Hydrogen peroxide (H₂O₂), a major photoproduct, induced SCE but failed to increase chromosome aberrations. Pure H₂O₂, or the H₂O₂ generated in light-exposed medium, was necessary and sufficient for inducing all the increase in SCE. However, H₂O₂ was necessary but insufficient to cause most of the chromosome aberrations. Only when acting synergistically with other photoproducts did H₂O, induce extensive chromosome aberrations. The relatively high cell densities at near confluence levels used in these experiments were less sensitive to light-induced effects, nevertheless the entire light exposure dosage range effected photoproduct production adequate for inducing SCE and chromosome aberrations. Thus, mammalian tissue and cell culture media can receive sufficient dosage from fluorescent lights illuminating rooms and culture hoods for generation of photoproducts causing gross and insidious SCE and chromosome alterations.     

INTERACTIONS OF ASCORBATE AND CHELATED IRON IN A METHYLVIOLOGEN-MEDIATED MEHLER REACTION

Abstract— –In the light, isolated spinach thylakoids consumed O₂ in the presence of methylviologen, and ascorbate was found to interact with this reaction in various ways. Chelating-resin was used to remove metal impurities from the assay medium. Ascorbate diminished the H₂0₂ pool in resin-untreated solutions, while in resin-treated solutions ascorbate had no effect on H₂O₂ concentrations. A Fenton catalyst (Fe-EDTA) increased O₂ uptake in the presence of ascorbate and decreased the amount of O₂ recovered by catalase. Ascorbate tripled the rate of the methylviologen-mediated Mehler reaction, and the O₂ consumed was liberated to 50% of its original concentration by catalase. Superoxide dismutase reversed the effects of ascorbate on the Mehler reaction rates. These results indicate that ascorbate can stimulate Mehler reactions indirectly by promoting a Fenton-type reaction as well as stimulating Mehler reactions directly by reducing 2O₂^- to 2H₂O₂. The promotion of a Fenton-type reaction by ascorbate appears to be the cause of H₂O₂ depletion in resin-untreated solutions.     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.     

EFFECTS OF QUENCHING AGENTS ON THE PHOTODYNAMICALLY-INDUCED CHEMOTACTIC RESPONSE OF THE COLORLESS FLAGELLATE Polytomella magna

Abstract In the presence of the photosensitizer riboflavin at high fluence rates a photoproduct, most probably H₂O₂, is formed which causes negative phototaxis in the colorless flagellate Polytomella magna . The aim of this study was to find out whether H₂O₂ is produced in a type I or II reaction. As has been shown, ¹O₂ quenchers either do not influence the photodynamic action of riboflavin (furfuryl ethanol, DPBF, l -histidine, crocetin) or show slight quenching effects only at very high concentrations ≧ 10⁻² M (DABCO, DMF, imidazole). D₂O is toxic to P. magna even in 1:1 and 1:2 mixtures with H₂O. On the other hand, the quenching effect of 1,4-benzoquinone, highly indicative for the type I pathway, is more than two orders of magnitude stronger than the one of the above mentioned ¹O₂ quenchers. The results suggest that H₂O₂ is produced in a type I reaction. Superoxide does not seem to be involved since superoxide dismutase does not diminish the photodynamic effect of riboflavin.     

NEAR-UV-INDUCED BREAKS IN PHAGE DNA: SENSITIZATION BY HYDROGEN PEROXIDE (A TRYPTOPHAN PHOTOPRODUCT)

Abstract— Near-UV irradiation of l -tryptophan yields a large number of photoproducts. When this mixture is added to recombinationless ( rec ) mutants of bacteria, the cells are killed. The most toxic component of tryptophan photoproducts has been identified as hydrogen peroxide (H₂O₂). We now report that both tryptophan photoproducts and H₂O₂ sensitize phage DNA to near-UV radiation resulting in enhanced killing as well as enhanced DNA breakages. We conclude that the in situ production of H₂O₂ via tryptophan photolysis may be an important biological event.     

ON THE GENERATION OF THE HYDROXYLATION AGENT FROM SUPEROXIDE RADICAL. CAN THE HABER–WEISS REACTION BE THE SOURCE OF OH RADICALS?

Abstract— In many biological systems, the role of O₂^- in hydroxylation and toxic processes was assumed to be due to the formation of OH radicals. The Haber-Weiss reaction (Haber and Weiss, 1934)—(H₂O₂+ O₂^-→ OH + OH^-+ O₂) was suggested as the origin of this activity.
In this study it is shown that this reaction pathway is too slow, and that OH is probably formed from the reaction of complexed superoxide with H₂O₂ or/and from the reduction of Fe(III), bound to biological compounds, by O₂^-; the reduced Fe(II) can then react with H₂O₂ as a Fenton reagent, to yield OH.
It is also shown that singlet oxygen cannot be formed in these biological systems neither from the dismutation of OJ nor from the reaction of O₂^- with OH. Singlet oxygen may be formed from the reduction of metal complexes by O₂^-.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

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.     

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.