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Mitochondrien aus Rattenleber (RL) und Rinderherzmuskel (BH) erzeugen bei Behandlung mit O2 eine schwache Chemilumineszenz in dem Spektralbereich zwischen 400 und 650 mµ, deren Intensität bei RL-Mitochondrien durch vorheriges Einfrieren und Auftauen wie durch Ultraschallbehandlung größer wird. Bei beiden Arten verursacht Zusatz von Acridinorange eine wesentliche Verstärkung der Lumineszenz, gleichzeitig wird die O2-Aufnahme gehemmt. RL-Mitochondrien erzeugen unter diesen Bedingungen eine kurzzeitige, BH-Mitochondrien eine langsam ansteigende langandauernde Strahlung; das gleiche Verhalten zeigen aus BH-Mitochondrien gewonnene „electrontransfer-particles“ (ETP). Der zeitliche Ablauf und die Beeinflußbarkeit durch Effektoren der Atmungskette ist andersartig als bei der von VLADIMIROV gefundenen Chemilumineszenz von RL-Mitochondrien. Als Träger der Lumineszenz wird angeregter O2 diskutiert.
In order to determine the influence of OH and O2H-radicals on proteins, bovine serum albumin (BSA) in aqueous solution was treated with Fenton’s reagent [Fe(II)SO4+EDTA+H2O2] and with ultraviolet light (λ > 2800 Å) in the presence of H2O2. The action of free radicals produced in this way did not change the properties of the native protein with respect to the sedimentation in the ultracentrifuge or optical rotatory dispersion and electrophoresis under normal conditions. Ampèrometric titration indicated partial oxidation of SH-groups and of 3—5 SS-groups which are not reducible by NaBH4.
Heat aggregation investigated by means of light-scattering was suppressed at pH 7.5 and strongly accelerated at pH 4.6 (range of coagulation), the latter being a result of increased entropy of activation of coagulation velocity.
The difference spectrum against native BSA had positive values of Δε and two maxima at 2480 and 2950 Å.
Ultracentrifugation at room temperature in phosphate buffer (pH 7.3, μ=0.18) furnishes a molecular weight of 63 300. In a solution of 8 M urea and borate buffer (pH 9, μ=0.05) fragments with molecular weights between 25 000 and 37 000 were observed while in phosphate buffer (pH 7.3, without urea) at temperatures higher than 46 °C an anomalous behaviour of the concentration gradient indicated an effect which possibly depends on a dissociation equilibrium.
As a consequence oxygen radicals seem to attack not only SH- and SS-groups but at least one covalent bond of the peptide chain. Some experiments of heat aggregation with BSA treated with γ-rays (60Co) gave the same results as BSA treated with Fenton’s reagent or UV-light+H2O2.
Diluted aqueous solutions of some proteins (bovine serum albumin, β-Lactoglobubin, Peroxidase) show weak phosphorescence lasting over several minutes after they have been irradiated with light in the range 3500-4200 A. Addition of Eosin after the irradiation amplifies in some cases the intensity of luminescence to a value of about hundred. If Eosin is present at the irradiation process the excitation to phosphorescence is possible with light of the wavelength 5460 A.
After denaturation processes which destroy the configuration of proteins (Urea, Guanidine-HCI. detergents, heat at higher pH) the ability of phosphorescence disappears altogether; likewise after blocking the SH-groups by benzochinone or a total oxidation or reduction of the SS-groups which causes an complete unfolding of the peptide chain.
In solutions of bovine serum-albumin irradiated with 3650 Å at room temperature and afterwards frozen to -178°C no radicals could be observed by measurements of electron-spin-resonance but they were detectable if the irradiation took place in the presence of H2O2.
The reactions Xanthinoxidase-Xanthine-O2, Peroxidase-H2O2 and bovine serum-albumin-H2O2-Fe (II) EDTA are accompanied by chemiluminescence. By comparison with the behaviour of oxidised serum-albumin it could be shown that the chemical reaction produces an excited state of the native protein.
The observations lead to the conclusion that the weak phosphorescence of long duration originates from a triplet-state which is sufficiently populated only as the consequence of cooperative phenomena attending the undisturbed α-Helix-structure of the protein.
During photooxidation of polycyclic aromatic hydrocarbons (PAH) products can be formed which develop chemiluminescence on treatment with bases. Flash photolysis experiments show that this is the case only after previous formation of cation radicals, e.g. in the presence of CCl4 as solvent or of e-acceptors in aprotic solvents. These radicals react with oxygen to peroxy-radicals which can combine to several kinds of peroxides. Primary and secondary peroxides are the sources of chemiluminescent activity.
Chemiluminescent peroxides can also be obtained by irradiation of PA H carbonyl com pounds in protic solvents under nitrogen. It is assumed that two excited CO groups combine exceptionally with their O-atom s thus creating a peroxide bond. 24 aromatic aldehydes, ketones, dicarboxylic acid anhydrides and coumarines develop chemiluminescence after illumination with wavelengths ≥ 320 nm with intensities varying 4 magnitudes of order.
The sensitivity of the photochemiluminescent method is sufficient to detect amounts of PA H and their CO derivatives in the ppb to ppm range.
The thermal decomposition of 1,2-diadamantyldioxetane was studied by kinetic and spectroscopic methods. Spectra of the chemiluminescence emitted during the thermally induced decomposition of 1,2-diadamantyldioxetane, tetramethyldioxetane and trimethyldioxetane were obtained and the influence of quenchers and radical-scavengers, and the presence of "heavy atoms" in the surrounding of the emitting species was investigated. The kinetics of the decay mechanism was followed by measuring the time dependence of the chemiluminescence. The influence of radical-scavengers, quenchers and "external heavy atoms" on the kinetics was assessed. Experimental results were discussed in terms of a biradical decay mechanism.
Durch fluoreszenz-spektrographische Intensitätsmessungen mit einem aus Laboratoriumsmitteln gebauten, einfachen Fluoreszenzspektrometer wurde die Löslichkeit des carcinogenen Kohlenwasserstoffs 3.4-Benzpyren in verdünnten, wäßrigen Lösungen von β-Lactoglobulin und Milchsäure-Dehydro-genase bestimmt. Die molare Lösungsvermittlung der Proteine für 3.4-Benzpyren ist erheblich größer als die des Koffeins. Die Fluoreszenz des 3.4-Benzpyrens wird in den Proteinlösungen nicht durch molekularen Sauerstoff gelöscht. Das beobachtete Spektrum gleicht demjenigen alkoholischer Lösungen von 3.4-Benzpyren ohne Konzentrationslöschung.
Um den Mechanismus der Reaktion des Cysteins mit molekularem Sauerstoff in Gegenwart von Komplexen des zweiwertigen Kupfers als Katalysatoren zu ermitteln, wurden Messungen der Chemilumineszenz, der Sauerstoff- und der Cysteinkonzentration in Abhängigkeit von der Zeit vorgenommen. Variation der Konzentration der Reaktionsteilnehmer führte zu Meßergebnissen, die die Aufstellung eines Reaktionsschemas gestattete. Das hieraus abzuleitende System nichtlinearer Differentialgleichungen für die Reaktionsgeschwindigkeiten wurde in einem Analogrechner gelöst, wobei Übereinstimmung zwischen Rechnung und Meßergebnissen sowohl für die Zeitabhängigkeit als auch für die Konzentrationsabhängigkeit gefunden wurde.
The autoxidation of NaSH and Cysteine in the presence of heavy metal ions is accompanied by chemiluminescence due to the formation of O2⊖ or adequate compounds as intermediates. The observation of the luminescence intensity and its time dependence has been used as analytical indication of the occurrence of electron transfer reactions from - SH to O2.
This enabled the study of the influence of different catalytic promoters. The efficiency of different metal ions could easily be demonstrated by their enhancement of light production during the reaction of NaSH with molecular oxygen. Cu (II) as one of the most efficient catalysts was also applied in the form of different complexes. Because it would catalyse the oxidation of cysteine, glutathione and other electron donors of biological interest, the influence of the nature of the ligands of the complexes was investigated. In the case of cysteine only complexes with stability constants of medium strength and planar configuration acted as effective catalysts. Therefore it has to be assumed that for an effective electron transport to the loosely bound oxygen the cysteine molecule has to enter the inner sphere of the complex. The much longer time of luminescence of this reaction (4 -10 min) compared to the short time luminescence caused by free O2H. OH and H2O2 indicates that these intermediates are stabilized by binding to the Cu (II) -complex as in compounds (I), (II) and (III) of oxidizing enzymes.
Lumineszenz von Hefe
(1968)
The reactions of diluted aqueous solutions of SO2 resp. HSO3-ions with MnO4-or Ce4+ ions in the pH range 1-4 produce chemiluminescence in the spectral region of 450-600 nm. Measurements of the time course of the light emission and their simulation on an analog computer led to a reaction scheme in which a recombination product of primarily formed HSO3 radicals -of a lifetime of about 1 second -appears as precursor of electronically excited SO2 molecules. The participation of singlet oxygen can be excluded because at least the reaction with Ce4+ ions proceeds also in the absence of oxygen.
Diadamantyldioxetane, trim ethyldioxetane and tetram ethyldioxetane were photolyzed b y light of A > 260 nm . The spectral distribution o f the quanta emitted during photoinduced decom position of dioxatenes was found to be different from fluorescence and phosphorescence o f ketones. Flash photolysis experim ents showed the absorption of an short-lived interm ediate. It was concluded, therefore, that photolysis o fdioxetanes is not a concerted process but involves at least one precursor o f the final product ketone.
In systems containing singlet-oxygen and aromatic fluorescers energy transfer from singletoxygen dimers to the dye should be observable by emission of the fluorescer. In order to prove this hypothesis, externally generated singlet-oxygen (1Δg) was bubbled through the solutions of dyes (chlorophyll a, eosin y, rhodamine b, luminol, rubrene and acridine orange) in organic solvents.
Luminescence could be observed and its spectral distribution analyzed by sharp cut-off filters and interference filters (rubrene) . Spectra, rates of oxidation, addition of quenchers and the long lasting time dependence of the reported reactions lead to the conclusion that the observed afterglow is due to chemical oxidation mechanisms producing a chemiluminescence. Therefore an excitation of the substances investigated in these experiments by simple physical energy transfer seems not to be predominant.
Singlet oxygen (1Δg) was generated by a microwave discharge and bubbled through a solution of chlorophyll-a in dibutylphtalate at approximately 10-20 torr. It not only excited the dye to its first singlet state but also produced oxidized species which generated a very long lasting weak chemiluminescence. From quenching experiments for the generation of the excited species could computer simulation.
The carcinogenic hydrocarbon 3.4-benzopyrene is soluble in aqueous solutions of different proteins. The solubilities are easily determined by the fluorimetric method. The fluorescence o. the hydrocarbon in the protein solutions is not quenched by molecular oxygen. Nevertheless only in presence of air (oxygen) an irreversible decrease of the fluorescence intensity occurs under irradiation with UV-light of wavelength 366 mμ, which is considerably faster than under nitrogen or in solutions of the hydrocarbon in ethanol or aqueous caffeine.
In the systems investigad, a correlation was found between the half-life period of the reaction and the SH-group activities. The participation of protein-SH-Groups in the 3.4-benzopyrene photoreaction is demonstrated by ampèrometric Ag⊕-titrations.
The influence of protein denaturation and inhibiting additives on the photoreaction are investigated by the fluorimetric method.
Irradiation- and oxygen-dependence of the reaction are analogous to the observations of photodynamic action and skin cancer induction by 3.4-benzopyrene.
By 366 mµ irradiation of β-lactoglobuline solutions containing 3.4-benzopyrene the heatdenaturation characteristics of the protein are changed. The same changes are produced without 3.4-benzopyrene by UV-light of the wavelength 280 mµ. Treatment of the β-lactoglobuline solutions with an amount of cigarette smoke, which certainly does not contain 3.4-benzopyrene in sufficient concentration, acts in the same direction.
Along with the changes in the protein properties the typical fluorescence of 3.4-benzopyrene vanishes. The hydrocarbon does not act as a catalyst in photodynamic action, but is chemically altered as well as the protein, at least in the system under investigation.
Exposite produce chemiluminescence when heated to 50 - 70 °C or treated with nucleophilic substances at room temperature. Initiation by Piperidine in Dimethylsulfoxide allows to determine 5 nmol of Phenyloxirane in 5 ml samples.
Interactions of eosin with three different substrates, β-lactoglobuline, bovine serum albumin and cysteine, in aqueous solutions of pH 7 under illumination with light of wavelengths 5200—5400 Å are investigated by changes in absorption spectrum characteristics, SH-group activities and phosphorescence intensities.
Only with bovine serum albumin the major part of protein conversion, as shown by spectral changes and diminution of SH-groups due to eosin-sensitized photo-oxidation. In β-lactoglobuline an oxidizing photoreaction occurs, by which eosin is vanishing to the same degree as the protein shows loss of SH-groups and spectral alterations indicating attack on aromatic amino acid residues. There is no red shift of the eosin absorption band at 5170 Å as is observed in solutions of bovine serum albumin, where the intensity of phosphorscence is about 100 fold compared with the intensity obtained by solutions of β-lactoglobulin.
The aerobic eosin photoreaction in solutions of β-lactoglobulin is faster than aerobic photobleaching of the dye. Still faster is its bleaching photoreaction with cysteine, which is nearly independent of oxygen.