Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe.

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)

K033-F1 2 x 96 well plate 425 EUR

OKAU00030-2PLATE - Catalase Fluorescent Activity Kit

OKAU00030-2PLATE 2plate 379 EUR

Amplite® Fluorimetric Catalase Assay Kit *Red Fluorescence*

11306-200Tests 200 Tests 222 EUR

Fluorescent

FPAK-3058--9K 9X1 mL 675.6 EUR

Fluoro Catalase

FLOCAT100-3 500 Tests 330 EUR

Fluorescent Particles

FH-10056-10 10 mL 1022.4 EUR

Fluorescent Particles

FH-3056-10 10 mL 802.8 EUR

Fluorescent Particles

FP-2054-2 2 mL 230.4 EUR

Fluorescent Particles

FP-10056-10 10 mL 888 EUR

Fluorescent Particles

FP-10056-2A 2X1 mL 310.8 EUR

Fluorescent PAK Blue

FP-0567-2 2 mL 218.4 EUR

LAMP Fluorescent Dye

RP001-01 100 μl 15.5 EUR

Fluorescent Agent-5

F597823 10mg 334 EUR

Lamp Fluorescent Tube

LAM3218 EACH 12.95 EUR

Fluorescent sensor, ANQ

9695-25 each 810 EUR

Fluorescent sensor, ANQ

9695-5 each 248.4 EUR

Catestatin (Human) - Fluorescent EIA Kit

FEK-053-27 96 wells 660.96 EUR

Fluorescent Protein Set

K816-6-100 each 1488 EUR

Fluorescent 100 bp DNA Ladder

M-234L 5 x 500µl 288.4 EUR

Fluorescent 100 bp DNA Ladder

M-234S 500µl 72.1 EUR

Fluorescent 200 bp DNA Ladder

M-235L 5 x 500µl 276.9 EUR

Fluorescent 200 bp DNA Ladder

M-235S 500µl 69.2 EUR

Fluorescent 500 bp DNA Ladder

M-236L 5 x 500µl 207.7 EUR

Fluorescent 500 bp DNA Ladder

M-236S 500µl 52 EUR

Cyan Fluorescent Protein

30R-2808 100 ug 462 EUR

Fluorescent Particle Kit

FX-3552-6K 6x1 mL 432 EUR

Fluorescent Particle Kit

FX-3552-9K 9X1 mL 675.6 EUR

Fluorescent Particle Kit

FA-2552-6K 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.

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