Hydrogen Peroxide Detection - New Trinder's Reagent

Peroxidase Detection

HPPA

SAT 3 / SAT-Blue

TMBZ / TMBZ-HCl / TMBZ-PS

Hydrogen Peroxide Detection

New Trinder’s Reagents, which are highly water-soluble aniline derivatives, are widely used in diagnostic assays and biochemical examinations. They have several advantages over conventional chromogenic reagents in the colorimetric determination of hydrogen peroxide activity. New Trinder’s Reagents are stable enough to use in both solution and test strip detection systems. New Trinder’s reagents form highly stable purple or blue dyes through an oxidative coupling reaction with 4-Aminoantipyrine (4-AA) or 3-Methylbenzothiazolinone hydrazone (MBTH) in the presence of hydrogen peroxide and peroxidase. MBTH has 1.5 to 2 times the molar absorptivity of 4-AA; however, 4-AA solution is more stable than MBTH solution. Enzymatic oxidization of a substrate by its oxidase produces hydrogen peroxide. The hydrogen peroxide concentration corresponds to the substrate concentration. Therefore, the amount of the substrate can be determined by the color development of an oxidative coupling reaction. Glucose, alcohol, acyl-CoA, and cholesterol are utilized for the detection of those substrates coupled with New Trinder’s Reagents and 4-AA. There are 10 kinds of New Trinder’s reagents available. The table on the next page shows the maximum wavelength and molar absorptivity of each oxidized New Trinder’s Reagent complex with 4-AA. Among the New Trinder’s reagents, TOOS is the most frequently used to develop assay systems. However, experimentation with the different New Trinder’s reagents will be necessary to develop the best detection system for a given substrate.

Perparation of Assay Solution - TOOS
1. Dissolve 20 mg TOOS with 10 ml PBS to prepare 6.6 mM TOOS solution.
2. Dissolve 14 mg 4-aminoantipyrin (AA) with 10 ml PBS to prepare 6.6 mM 4-AA solution.
3. Prepare 2 U/ml horseradish peroxidase solution with PBS.
4. Mix the same volume of each solution together to prepare assay solution. Store the assay solution at 4 ºC with protection from light.

Assay Protocol
1. Prepare sample solutions for the enzymatic oxidation reaction. The pH range of the buffer solution should be from 5.5-9.5.
2. Prepare standard solutions containing known amounts of substrate using the same buffer.
3. Add the appropriate units of oxidase to the sample solution, followed by addition of the same volume of the assay solution.
4. Incubate the mixture at room temperature or at 37 ºC for 30 min to 1 hour.
5. Measure the O.D. at 555 nm.
6. Prepare a standard curve and determine the substrate concentration in the sample solution.

Product Name: ADOS N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxylaniline, sodium salt, dihydrate

Product Code: OC01-10    Unit: 1 g

Product Code: OC01-12    Unit: 5 g

Oxidation Reaction

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Product Name: ADPS N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline, sodium salt, monohydrate

Product Code: OC02-10    Unit: 1 g

Product Code: OC02-12    Unit: 5 g

Oxidation Reaction

Product Name: ALPS N-Ethyl-N-(3-sulfopropyl)aniline, sodium salt

Product Code: OC04-10   Unit: 1 g

Product Code: OC04-12    Unit: 5 g

Oxidation Reaction

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Product Name: DAOS N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, sodium salt

Product Code: OC06-10   Unit: 1 g

Product Code: OC06-12   Unit: 5 g

Oxidation Reaction

Product Name: HDAOS N-(2-Hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, sodium salt

Product Code: OC08-10    Unit: 1 g

Product Code: OC08-12    Unit: 5 g

Oxidation Reaction

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Product Name: MADB N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline,disodium salt

Product Code: OC21-10    Unit: 1 g

Product Code: OC21-12    Unit: 5 g

Oxidation Reaction

Product Name: MAOS N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, sodium salt, monohydrate

Product Code: OC11-10    Unit: 1 g

Oxidation Reaction

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Product Name: TODB N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt

Product Code: OC22-10     Unit: 1 g

Product Code: OC22-12     Unit: 5 g

Oxidation Reaction

Product Name: TOOS N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate

Product Code: OC13-10    Unit: 1 g

Product Code: OC13-12    Unit: 5 g

Oxidation Reaction

 

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Product Name: TOPS N-Ethyl-N-(3-sulfopropyl)-3-methylaniline, sodium salt

Product Code: OC14-10    Unit: 1 g

Product Code: OC14-12    Unit: 5 g

Oxidation Reaction

Peroxidase Assay

Product Name: HPPA

Description:

Appearance: white powder
Purity: >99.0 % (Tit.)
m.p.: 127-134 ºC
Solubility: 100 mg/10 ml methanol

Oxidation Reaction
HPPA is one of the fluorescent reagents used to determine the peroxidase and hydrogen peroxide activities. It produces fluorescent dimers and trimers in the presence of peroxidase and hydrogen peroxide. The propionic acid analog of hydroxyphenyl is more sensitive than the acetic acid analog. This reagent is also more sensitive than homovanilic acid or p-hydroxyphenylacetic acid: Sub-femtomolar levels of peroxidase can be detected with HPPA. HPPA solution can be stored at -20 ºC for several months. The oxidized HPPA is also stable at room temperature. Dojindo offers carefully manufactured HPPA that has a very high signal-to-noise ratio. The excitation and emission wavelengths of oxidized HPPA are 330 nm and 410 nm, respectively.

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Product Name: SAT 3

Description:

Appearance: white or slightly brown powder
Purity: >93.0 % (HPLC)
m.p.: 220-251 ºC (dec.)
Molar absorptivity: >43,000 (213 nm)
Solubility: 16 mg/ml water
Water content: <6 %/ pH: 7.2-8.2 (0.5 % solution)

SAT-3 is a stable, highly water-soluble o-Tolidine analog. It is easily oxidized by peroxidase and hydrogen peroxide into a green dye at pH 4-6. While TMBZ requires an organic solvent or detergent to solubilize, SAT-3 can be solubilized with just buffer solution. The SAT-3 solution can be stored at room temperature with protection from light. The maximum wavelength of the dye is 675 nm.? No mutagenicity of SAT-3 was detected using the Ames test.

Assay Protocol
1. Mix 1 ml of 100 mM SAT-3 solution and 1 ml of 30 mM hydrogen peroxide solution with 8 ml of 50 mM citrate buffer (pH 4) to prepare an assay solution.
2. Add 100-200 µl of the assay solution to each well of a 96-well plate and incubate at 37 ºC for 5-30 min.
3. Read the O.D. at 670 nm or add 50 ml of 4 M sulfuric acid to each well and read the O.D. at 490 nm instead.

Product Name: SAT Blue

Description:

Appearance: white or slightly brown color solution
Purity: >93.0 % (HPLC)
m.p.: 220-251 ºC (dec.)
Molar absorptivity: >43,000 (213 nm)
Water content: <6 %/ pH: 7.2-8.2 (0.5% solution)

Oxidation Reaction


SAT Blue is a ready-to-use solution of SAT-3 and other ingredients. SAT Blue contains no organic solvents. SAT Blue produces an intense green dye if it is mixed with peroxidase. The maximum wavelength of oxidized SAT Blue is 675 nm. SAT Blue is suitable for a microplate enzyme immunoassay based on the peroxidase activity detection mechanism. Like TMBZ, oxidized SAT-3 forms a yellow dye by the addition of 2 M sulfuric acid.

Assay Protocol
1. Prepare an assay plate with peroxidase activity using a peroxidase-labeled antibody or peroxidase-labeled streptavidine.
2. Wash each well 4-5 times with 200-300 ml PBST (0.05% Tween 20 PBS buffer).
3. Add 100-200 µl SAT-Blue solution to each well and incubate at room temperature or 37 ºC for 15-30 min.
4. Measure the O.D. at 650 nm or 670 nm.

Absorption Spectrum of Oxidized Form

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Product Name: TMBZ

Description:

Appearance: white or pale grayish brown crystalline powder
Purity: >99.0 % (HPLC)
m.p.: 165-171 ºC
Molar absorptivity: >24,000 (287 nm)
Solubility: 100 mg/10 ml hot methanol

Oxidation Reaction


TMBZ is a chromogenic reagent utilized for peroxidase detection. It has been developed as an alternative to benzidine, which is a carcinogenic chemical. Because of the ortho methyl groups on its benzene ring, TMBZ is not metabolized into highly carcinogenic o-hydroxybenzidines or o,o·dihydroxybenzidines. Therefore, TMBZ compounds are much less carcinogenic than benzidine. Though the TMBZ solution is colorless, it turns bluish-green (lmax: 655 nm) in the presence of hydrogen peroxide and peroxidase. The structure of this bluish-green complex is thought to be a radical form of two oxidized TMBZ molecules. TMBZ HCl is a hydrochloride form of TMBZ that is readily soluble in water (100 mg TMBZ HCl per 10 ml water). TMBZ-PS is a water-soluble analogue (50 mg TMBZ-PS per 10 ml water) of TMBZ that has a sulfate group.

Assay Protocol
1. Dissolve 6 mg of TMBZ with 1 ml DMSO to prepare 100X TMBZ solution.
2. Mix 5 µl of 30% hydrogen peroxide solution with 1 ml PBS to prepare 200X H₂O₂ solution.
3. Add 10 µl of 100X TMBZ solution and 5 µl of 200X H₂O₂ solution to 1 ml PBS to prepare staining solution.^a)
4. Add 100-200 µl of the staining solution to each well of a 96-well microplate. Incubate the plate at room temperature or at 37 ºC for 5 min to 1 hour.
5. Wash the sample with PBS several times to stop the staining reaction.

   a) For the best result, modification of the final concentration of TMBZ and hydrogen peroxide may be necessary. The staining solution is not stable. Please prepare fresh solution prior to use.

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Product Name: TMBZ HCl

Description:

Appearance: white or slightly pink crystalline powder
Purity (as dihydrate): >98.0 % (Tit.)
Water content: 8-12 %/ pH: 2.0-2.5
Solubility: 100 mg/10 ml water

Oxidation Reaction

Product Name: TMBZ-PS

Description:

Appearance: white or pale yellow powder
Purity: >93.0 % (HPLC)
m.p.: 220-251 ºC (dec.)
Molar absorptivity: >43,000 (213 nm)
Solubility: 50 mg/10 ml water
Water content: <6 %/ pH: 7.2-8.2 (0.5 % solution)
Licensed under U.S. Patent, No. 4,716,110 Oxidation Reaction

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