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1.
Bioluminescent oxidation of Cypridina luciferin yields CO2 besides oxyluciferin and light. The exchange of oxygen between the CO2 and H2O of the solvent becomes significant when less than approximately 1 μmol of luciferin is reacted in 4 ml of buffer solution, and the exchanged oxygen in CO2 markedly increases by decreasing the amount of luciferin. Such an exchange is to be expected in any such system which produces CO2 in aqueous solution, and must be taken into account in interpreting the results of experiments.  相似文献   

2.
The use of 14C-carboxyl-labelled luciferin as a substrate for the firefly luciferase catalyzed reaction produces 14CO2 as a product. We have studied this reaction in the presence of 17O2 and H18OH, using an excess of luciferin over luciferase. The initial collection of CO2 contained close to one oxygen from 17O2 for each molecule of 14CO2 derived from luciferin, which is consistent with a cyclic peroxide mechanism. About half of the 14CO2 remained bound to the enzyme and was collected after acidification of the medium. This CO2 contained less than 0.1 of an atom of oxygen from 17O2 for each molecule of 14CO2 derived from luciferin. Exchange of medium CO2-HCO3 su? with water was not sufficiently great to account for the loss of any 17O previously incorporated. The most likely explanation appears to be a preferential exchange of oxygens of enzyme-bound CO2 with water oxygens. Such exchange, and dilution of CO2 from luciferin by medium CO2, may explain previous results in which little incorporation of atmospheric oxygen was noted.  相似文献   

3.
Mass spectral analyses of the CO2 liberated in the Cypridina luciferin-luciferase and firefly luciferin-luciferase reactions run in the presence of 17O2 and H218O show that the product is predominantly C18O16O (mass 46) and not C17O16O (mass 45). Incorporation of 18O into medium CO2 by exchange does not account for the observed results. These experiments provide evidence that the Cypridina and firefly bioluminescence reactions proceed via a linear peroxide mechanism rather than the dioxetane mechanism and suggest that a common mechanism may underly many bioluminescence reactions.  相似文献   

4.
A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O2 = oxyluciferin + H2O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.  相似文献   

5.
The use of a fully active, synthetic analogue of coelenterate-type luciferin labeled in the carbonyl position with 14C and 18O was used to probe the mechanism of the Renilla luciferase catalyzed oxidative decarboxylation of this compound. In the presence of 17O2, the CO2 produced in this oxidation can be shown to contain approximately one 17O atom per CO2 molecule. This result is consistent with a cyclic peroxide or dioxetanone-type mechanism. In the presence of luciferase, the oxygen in the luciferin carbonyl group is rapidly exchanged with solvent water prior to the production of CO2. Thus, the reaction CO2 contains considerable oxygen derived from water, via exchange with the carbonyl group, and about one oxygen from O2 via a cyclic peroxide.  相似文献   

6.
Reactive oxygen species (ROS) are presently thought to play important role in an increasing number of the physiological and pathological processes in living organisms. Various chemiluminescent (CL) compounds have been studied in order to find suitable and specific probes for the detection of particular ROS species. The CL of luminol is known to be non‐specific and can be induced by various oxidants. Two Cypridina luciferin analogues, CLA and MCLA, have been used for the detection of ROS in vivo. CLAs are thought to emit light only when reacting with superoxide and singlet oxygen. It is possible to distinguish the particular ROS by using a specific quencher or scavenger, e.g. superoxide dismutase (SOD) or sodium azide (NaN3). The CL reactions of luminol (3‐aminophthalhydrazide), CLA [2‐methyl‐6‐phenyl‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] and MCLA [2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] were studied in three hydrogen peroxide decomposition systems (H2O2–HRP; H2O2–CuSO4; and H2O2–NaOCl). The measurements were carried out in phosphate buffer, pH 7.4, at 25 °C, using a luminometer (Fluoroskan Ascent FL and Sirius C). NaN3 was used as the specific quencher of singlet oxygen. The results demonstrate that the proclaimed specifity of the CL of Cypridina luciferin analogues towards singlet oxygen has to be discussed. Copyright © 2011 John Wiley & Sons, Ltd.  相似文献   

7.
The unicellular green alga Chlamydomonas reinhardtii possesses a CO2-concentrating mechanism. In order to measure the CO2 permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO2 · L?1} (high-Ci cells) or in ambient air (350 μL CO2 · L?1}; low-Ci cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-Ci cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [14C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-Ci cells of C. reinhardtii were used to measure the exchange of 18O between doubly labelled CO2 (13C18O2) and H2O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the 18O depletion from 13C18O2 in the external medium provides a powerful tool to study CO2 diffusion across the PM to the active site of CA which catalyses 18O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the 18O loss to H2O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the 18O-depletion kinetics performed at pH 7.3 and 7.8, CO2 permeability coefficients of 0.76 and 1.49·10?3} cm·s?1}, respectively, were calculated for high Ci cells. The corresponding values for low-Ci cells were 1.21 and 1.8·10?3} cm·s?1}. The implications of the similar and rather high CO2 permeability coefficients (low CO2 resistance) in high and low-Ci cells for the COi-concentrating mechanism of C. reinhardtii are discussed.  相似文献   

8.
A nonspecific liver esterase was found to not only catalyze the hydrolysis of the methyl ester of prostaglandin F2α (PGF2α) but also to catalyze the exchange of the carboxyl oxygen atoms with water leading to the production of [18O2]PGF2α when the enzymatic hydrolysis is carried out in H218O. The kinetics of this oxygen-18 exchange reaction are briefly discussed. The [18O2]PGF2α was found to be relative stable toward back exchange in methanol, aqueous buffer, and urine, but rapidly back exchanged to the native PGF2α in plasma with a half-life of 1 h. The [18O2]PGF2α was relatively stable in plasma to which alcohol had been added. The utility of the oxygen-18 labeled prostaglandin as an internal standard in a gas chromatography-mass spectrometry assay was demonstrated at the picomole range.  相似文献   

9.
The 18O-enrichment of CO2 produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular 18O2. The only 18O-labeled species produced was C18O16O; no C18O18O could be detected. Production of C18O16O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO2. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O2 present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO2 and H2O, drastically diminished the 18O-enrichment of the produced CO2. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the 18O labeling of photorespiratory CO2 could be observed for the first time. These data allow the establishment of a direct link between O2 consumption and CO2 production in the light, and the conclusion that CO2 produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C18O16O and CO2 kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO2 production in the light.  相似文献   

10.
β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either 16O2-H218O or 18O2-H216O medium for 15 min at 37 °C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from 18O-retinal-H216O and 16O-retinal-H218O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.  相似文献   

11.
《BBA》2019,1860(12):148060
Oxygen (O2) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O2 cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only 57Fe(II) or Mn(II) plus 57Fe(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including 16/18O and H/D exchange. 57Fe-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mössbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H2O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (μOH) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas 18O from 18O2 cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O2 reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O2 cleavage route in an enzyme.  相似文献   

12.
Measurements were made of the concentration and stable oxygen isotopic ratio of carbon dioxide in air samples collected on a diurnal basis at two heights within a Pinus resinosa canopy. Large changes in CO2 concentration and isotopic composition were observed during diurnal time courses on all three symple dates. In addition, there was strong vertical stratification in the forest canopy, with higher CO2 concentrations and more negative 18O values observed closer to the soil surface. The observed daily increases in 18O values of forest CO2 were dependent on relative humidity consistent with the modelled predictions of isotopic fractionation during photosynthetic gas exchange. During photosynthetic gas exchange, a portion of the CO2 that enters the leaf and equilibrates with leaf water is not fixed and diffuses back out of the leaf with an altered oxygen isotopic ratio. The oxygen isotope ratio of CO2 diffusing out of a leaf depends primarily on the 18O content of leaf water which changes in response to relative humidity. In contrast, soil respiration caused a decline in the 18O values of forest CO2 at night, because CO2 released from the soil has equilibrated with soil water which has a lower 18O content than leaf water. The observed relationship between diurnal changes in CO2 concentration and oxygen isotopic composition in the forest environment were consistent with a gas mixing model that considered the relative magnitudes of CO2 fluxes associated with photosynthesis, respiration and turbulent exchange between the forest and the bulk atmosphere.  相似文献   

13.
The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H2O2 (10mmol/L) and MCLA (10μmol/L) at pH 5.8.  相似文献   

14.
Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of 16O2, 18O2, H216O, and H218O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of 18O-compounds. Incorporation of one 18O atom in ODTD was found if the cleavage reaction was performed in the presence of 18O2 and H216O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H218O in the presence of 16O2 indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of 18O2, H216O, and alcohol dehydrogenase/NADH, incorporation of two atoms of 18O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.  相似文献   

15.
The oxygen stable isotope composition (δ18O) of CO2 is a valuable tool for studying the gas exchange between terrestrial ecosystems and the atmosphere. In the soil, it records the isotopic signal of water pools subjected to precipitation and evaporation events. The δ18O of the surface soil net CO2 flux is dominated by the physical processes of diffusion of CO2 into and out of the soil and the chemical reactions during CO2–H2O equilibration. Catalytic reactions by the enzyme carbonic anhydrase, reducing CO2 hydration times, have been proposed recently to explain field observations of the δ18O signatures of net soil CO2 fluxes. How important these catalytic reactions are for accurately predicting large‐scale biosphere fluxes and partitioning net ecosystem fluxes is currently uncertain because of the lack of field data. In this study, we determined the δ18O signatures of net soil CO2 fluxes from soil chamber measurements in a Mediterranean forest. Over the 3 days of measurements, the observed δ18O signatures of net soil CO2 fluxes became progressively enriched with a well‐characterized diurnal cycle. Model simulations indicated that the δ18O signatures recorded the interplay of two effects: (1) progressive enrichment of water in the upper soil by evaporation, and (2) catalytic acceleration of the isotopic exchange between CO2 and soil water, amplifying the contributions of ‘atmospheric invasion’ to net signatures. We conclude that there is a need for better understanding of the role of enzymatic reactions, and hence soil biology, in determining the contributions of soil fluxes to oxygen isotope signals in atmospheric CO2.  相似文献   

16.
Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO2 buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O2 outlet channel. Referred to surface area, rates of CO2 transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO2 supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L?1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L?1 was observed during repeated two-day cycles. Separate pathways for CO2 supply and oxygen outlet prevented significant loss of CO2. Convective gas flow through the oxygen outlet channel enabled the estimation of the O2 generation rate. The permeability of the channel for diffusive O2/N2 exchange limited the O2 concentration to a moderate value. It is concluded that shaken flat cultures using CO2 supply through a porous hydrophobic membrane and diffusive release of O2 through a separate pathway are promising for research on microalgae and cyanobacteria.  相似文献   

17.
The cyanobacterium Acaryochloris marina was cultured in the presence of either H218O or 18O2, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H218O, newly synthesized Chl a and d, both incorporated up to four isotopic 18O atoms. Time course H218O labeling experiments showed incorporation of isotopic 18O atoms originating from H218O into Chl a, with over 90% of Chl a 18O-labeled at 48 h. The incorporation of isotopic 18O atoms into Chl d upon incubation in H218O was slower compared with Chl a with ∼50% 18O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of 18O2 gas, one isotopic 18O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H218O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic 18O atoms derived from molecular oxygen (18O2) was observed in the extracted Chl d, and the percentage of double isotopic 18O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C31-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.  相似文献   

18.
Alpine treelines are temperature‐limited vegetation boundaries. Understanding the effects of elevated [CO2] and warming on CO2 and H2O gas exchange may help predict responses of treelines to global change. We measured needle gas exchange of Larix decidua Mill. and Pinus mugo ssp. uncinata DC trees after 9 years of free air CO2 enrichment (575 µmol mol?1) and 4 years of soil warming (+4 °C) and analysed δ13C and δ18O values of needles and tree rings. Tree needles under elevated [CO2] showed neither nitrogen limitation nor end‐product inhibition, and no down‐regulation of maximal photosynthetic rate (Amax) was found. Both tree species showed increased net photosynthetic rates (An) under elevated [CO2] (L. decidua: +39%; P. mugo: +35%). Stomatal conductance (gH2O) was insensitive to changes in [CO2], thus transpiration rates remained unchanged and intrinsic water‐use efficiency (iWUE) increased due to higher An. Soil warming affected neither An nor gH2O. Unresponsiveness of gH2O to [CO2] and warming was confirmed by δ18O needle and tree ring values. Consequently, under sufficient water supply, elevated [CO2] induced sustained enhancement in An and lead to increased C inputs into this ecosystem, while soil warming hardly affected gas exchange of L. decidua and P. mugo at the alpine treeline.  相似文献   

19.
Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H218O showed incorporation of up to three 18O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H2O. Toxin formed in the presence of 18O molecular oxygen incorporated up to six 18O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H218O and 18O2 labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.  相似文献   

20.
The occurrence of an active CO2 transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H13CO3? caused a rapid initial drop, followed by a slower decline in the extracellular CO2 concentration. Addition of bovine CA to the medium raised the CO2 concentration by restoring the HCO3?–CO2 equilibrium, indicating that cells were taking up CO2 and were maintaining the CO2 concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO2 concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of 18O from 13C18O2 into water. Exchange of 18O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO2 uptake by active transport for use as a carbon source for photosynthesis.  相似文献   

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