The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2).
These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ>> 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.
[Construction and functional identification of red fluorescent protein reporter plasmid for human catalase gene].
OBJECTIVE
To construct a red fluorescent protein reporter gene driven by human catalase gene promoter.
METHODS
The red fluorescent protein reporter gene plasmid pDsRed-CATp containing human catalase gene promoter was constructed by gene recombination technique. The plasmid was transiently transfected into NIH/3T3 cells to observe their response to H(2)O(2) stimulation.
RESULTS
The plasmid was constructed correctly as verified by double enzyme digestion and sequence analysis. The plasmid was lowly expressed in resting NIH/3T3 cells, but the expression level increased obviously after stimulation by H(2)O(2). CONCLSIONS: A red fluorescent protein reporter gene plasmid driven by human catalase gene promoter has been constructed successfully with a sensitive response to H(2)O(2) stimulation. This system provides a convenient tool for the study of the regulatory mechanism of catalase gene expression.
Catalase and superoxide dismutase of root-colonizing saprophytic fluorescent pseudomonads.
Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h, the cells displayed this low catalase but maintained high superoxide dismutase specific activities.
Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.
Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate.
A one-step method is described for the fluorometric determination of the activity of the enzyme catalase (EC 1.11.1.6.), based on the finding that H(2)O(2) in the europium (III)-tetracycline-hydrogen peroxide system is consumed by catalase. This is accompanied by a large decrease in both fluorescence intensity and decay time. The limit of detection (LOD; at S/N=3) for catalase at 30 degrees C for a 10-min kinetic assay is 1.0 unit/mL, with a linear range from 1.0 to 10 unit/mL. At an incubation time of 30 min at 37 degrees C for a one-point assay, the LOD is 0.046 unit/mL, with a linear range from 46 to 400 munit/mL.
The assay was performed on microtiterplates and is fully compatible with existing plate readers. It is a one-step, simple, and sensitive method suitable for both continuous kinetic and one-point detections, does not require the addition of other substrates, and works best at neutral pH (with an optimum at pH 6.9). The reagent has the typical spectral features of a europium-ligand complex including a large Stokes shift (210 nm), a red line-like emission (centered at 616 nm), and a decay time in the microsecond domain. It is also the first europium-based probe that is compatible with the 405-nm diode laser. In summary, the new assay provides distinct advantages over direct ultraviolet detection and over the two-reagent (peroxidase) method.
Catalase Fluorescent Activity Kit (2 Plate) |
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| K033-F1 | 2 x 96 well plate | 425 EUR |
OKAU00030-2PLATE - Catalase Fluorescent Activity Kit |
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| OKAU00030-2PLATE | 2plate | 379 EUR |
Amplite® Fluorimetric Catalase Assay Kit *Red Fluorescence* |
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| 11306-200Tests | 200 Tests | 222 EUR |
Recombinant Pseudomonas fluorescens Catalase-peroxidase (katG), partial |
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| MBS1309152-INQUIRE | INQUIRE | Ask for price |
Recombinant Pseudomonas fluorescens Catalase-peroxidase (katG), partial |
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| MBS1407407-INQUIRE | INQUIRE | Ask for price |
Fluorescent |
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| FPAK-3058--9K | 9X1 mL | 675.6 EUR |
Fluorescent DOTAP |
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| HY-143702 | 1 mg | 2380.99 EUR |
Fluorescent Particles |
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| FP-10056-10 | 10 mL | 888 EUR |
Fluorescent Particles |
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| FP-10056-2A | 2X1 mL | 310.8 EUR |
Fluorescent Particles |
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| FP-2054-2 | 2 mL | 230.4 EUR |
Fluorescent Particles |
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| FH-10056-10 | 10 mL | 1022.4 EUR |
Fluorescent Particles |
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| FH-3056-10 | 10 mL | 802.8 EUR |
Fluoro Catalase |
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| FLOCAT100-3 | 500 Tests | 330 EUR |
Fluorescent PAK Blue |
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| FP-0567-2 | 2 mL | 218.4 EUR |
LAMP Fluorescent Dye |
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| RP001-01 | 100 μl | 15.5 EUR |
LAMP Fluorescent Dye |
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| RP001-01-100ul | 100 μl | 15.81 EUR |
Fluorescent Agent-5 |
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| F597823 | 10mg | 334 EUR |
Lamp Fluorescent Tube |
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| LAM3218 | EACH | 12.95 EUR |
Fluorescent sensor, ANQ |
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| 9695-25 | each | 810 EUR |
Fluorescent sensor, ANQ |
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| 9695-5 | each | 248.4 EUR |
Catestatin (Human) - Fluorescent EIA Kit |
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| FEK-053-27 | 96 wells | 660.96 EUR |
Fluorescent Protein Set |
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| K816-6-100 | each | 1488 EUR |
Cyan Fluorescent Protein |
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| 4996-100 | each | 451.2 EUR |
Cyan Fluorescent Protein |
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| 4996-1000 | each | 2865.6 EUR |
Cyan Fluorescent Protein |
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| 4996-5000 | each | 7824 EUR |
Cyan Fluorescent Protein |
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| 30R-2808 | 100 ug | 462 EUR |
Fluorescent Particle Kit |
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| FA-2552-6K | 6x1 mL | 554.4 EUR |
Fluorescent Particle Kit |
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| FA-3558-6K | 6x1 mL | 554.4 EUR |
Fluorescent Se-modified carbon nitride nanosheets as biomimetic catalases for free-radical scavenging.
Efficient biomimetic catalases have been broadly explored for free-radical scavenging and antioxidative stress. Herein, we introduce a non-toxic Se-modified carbon nitride (CN) nanosheet for this bioapplication, and it presents efficient catalase activity, high fluorescence properties, and good biocompatibility. These properties indicate that this material has potential for use as an artificial enzyme against oxidative stress.