Peroxidases substrates

Akhavan-Tafti et al. have developed a new class of peroxidase substrates that produce CL upon enzymatic oxidation. Horseradish peroxidase (HRP) is... 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Reactivity of the Oxycomplex. The oxycomplex of lignin peroxidase does not react with veratryl alcohol, one of the lignin peroxidase substrates. Furthermore, the stability of the oxycomplex is not affected by veratryl... 

Culture media partially optimized for P. chrysosporium (50) unless otherwise indicated, nmol/min. measured at room temperature per mg dry mycelial weight for vanillylacetoneas a Mn -dependent peroxidase substrate and veratiyl alcohol as a lignin peroxidase substrate 0 denotes activity below detectable limit while — indicates assay not performed. Time, at 39 C for P. chrysosporium or at 30 C for L edodes, after inoculation when maximum activity appears (d ). Interval over which C02 evolution from dehydrogenative polymerizate (DHP) of [ring- /- C]conifeiyl alcohol is measured (days after inoculation). Culture media partially optimized for L. edodes at 22 C (83). 

The extent of inhibition of the oxidation of peroxidase substrates by ferulic salts was quite variable, from no inhibition to total inhibition. Total inhibition occurred when the substrate (e.g., syringaldazine) was closely related to ferulic acid. The presence of a fluorine atom in ferulic acid slightly reduced the inhibitory effect. Oxidation of ferulic compounds was restricted to lignifying cell walls in situ. Cell wall peroxidases from bark and xylem were fractionated into their component isozymes. 

Table I. Staining Relative Intensity Observed in Tobacco Stem Sections Incubated with Different Peroxidase Substrates. Transverse sections were incubated with H2O2 and a peroxidase substrate. The oxidation of routinely used commercial substrates was checked in the presence or the absence of ferulic acid salts. Variations of staining intensity in cell walls (— to ++) were judged by observation with a light microscope...

A new trend in the field of oxidations catalyzed by metalloporphyrin complexes is the use of these biomimetic catalysts on various supports ion-exchange resins, silica, alumina, zeolites or clays. Efficient supported metalloporphyrin catalysts have been developed for the oxidation of peroxidase-substrates, the epoxidation of olefins or the hydroxylation of alkanes. 

Substrate products can be classified as either soluble or precipitating. Soluble peroxidase substrates include o-phenylenediamine, which is converted into a yellow product 2,2 -azino-(3-ethyl)-benzothiazoline-sulfonic acid, which is converted into a green product and tetramethylbenzidine, which is converted into a blue product. Precipitating substrates for peroxidase include 4-chloronaphthol, which yields a blue precipitate and aminoethylcarbizole, which forms a red precipitate. Alkaline phosphatase is most frequently used with p-nitrophenyl phosphate to give a yellow-orange soluble product, or with 5-bromo-4-chloro-3-indo-lyl-phosphate p-toluidine salt to yield an insoluble blue product. 

Enzyme substrates- A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphosphatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl carbazole (AEC) are described. Other substrates can be found in textbooks of histochemistry (see also Chapter 24) a NBT/BCIP is made in advance, and stored at -20°C. After equilibration of 30 mL of alkaline phosphatase substrate buffer at 37°C, 10 mg of NBT are dissolved m 200 pL of dimethylformamide (DMF), and added to I mL of the prewarmed substrate buffer. The mixture is added dropwise to the remaining substrate buffer BCIP (5 mg), dissolved in 200 pL of DMF, is then added slowly, and the whole preparation stored m 4-mL aliquots at—20°C b AEC is prepared fresh daily by dissolving 2 mg of AEC in 1 2 mL of dimethyl-sulfoxide in a glass tube. The mixture is added to 10 mL of 20 mM acetate buffer, pH 5 0-5.2. Immediately prior to use, 1 pL of 30% (v/v) hydrogen peroxide is added The final mix may require filtration prior to use... 

Wash m TBS (5 min), and incubate in either alkaline phosphatase or peroxidase substrate. 

Peroxidase substrate (DAB). 2 5 mg of 3,3-diaminobenzidine tetrahydrochlonde dissolved m 5 mL phosphate-buffered saline (PBS). Add 2 pL of 30% hydrogen peroxide (H2O2). DAB powder should be handled only in a fume cupboard. 

One hundred microliters of commercial goat antirabbit globulin conjugated to horseradish peroxidase (diluted 1/500 diluting buffer) is added to every well. Incubation is done for 30 min at 37°C, followed by washing, and 100 pi of peroxidase substrate in freshly prepared substrate buffer is added to each well. 

Simultaneous detection of three antigens within one tissue section became possible by employing an additional peroxidase substrate such as the Vector VIP Substrate kit (Vector Lab, Burlingham, CA) (Pujic et al., 1998). This substrate is oxidized by horseradish peroxidase and yields a rose-colored final reaction product which differs in color from that... 

Petersen KH. Novel horseradish peroxidase substrates for use in immunohistochemistry. J Immunol Methods 2009 340(1) 86-9. 

Sundaramoorthy M, Youngs HL, Gold MH et al (2005) High-resolution crystal structure of manganese peroxidase substrate and inhibitor complexes. Biochemistry 44 6463-6470... 

The common overall reaction of the peroxidases can be written as in the following equation, where RH is a suitable peroxidase substrate and R is a free-radical product derived from it ... 

Camarero S, Sarkar S, Ruiz-Duenas FJ et al (1999) Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites. J Biol Chem 274 10324-10330... 

Add peroxidase substrate dissolved in acidic citrate/phosphate buffer and incubate 20 min (maximum) in the dark. 

Flood with peroxidase substrate solution (400 mg diaminobenzidine in 10 mL PBS, containing 0.01% hydrogen peroxide or Vector kit) for 10 min. 

Water beetle (Dytiscus) defence bombardier beetle (Bradynus) peroxidase substrate for ultimate benzoquinone discharge... 

Enzyme substrates. A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphos-phatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl car-bazole (AEG) are described. Other substrates can be found in textbooks of histochemistry (see a o Chapter 10). 

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