Solarbio Hydrogen Peroxide (H2O2) Content Assay Kit

Components:

Reagent I: Acetone 100mL×1. Storage at 4℃. (Self-provided reagent)

Reagent Ⅱ: Powder×1. Storage at 4℃. Working solution: Add 3mL concentrated hydrochloric acid (37%) before use, fully dissolved. Unused reagents stored at 4 °C.

Reagent Ⅲ: 6mL×1. Storage at 4℃.

Reagent Ⅳ: 30mL×1. Storage at 4℃.

Standard: 1mL×1, 1mmol/mL H2O2 standard solution. Storage at 4℃.

Product Description:

H2O2 is the most common reactive oxygen molecules in organisms. It is mainly produced by the catalyzation of SOD and XOD and degraded by the catalyzation of CAT and POD. H2O2, which is not only one of the important reactive oxygen, but also the hub of mutual conversion of reactive oxygen. On the one hand, H2O2 can directly or indirectly oxidize intracellular nucleic acids, proteins and other biological macromolecules, and damage cell membranes, thus accelerating the aging and disintegration of cells. On the other hand, H2O2  is also a key regulatory factor in many oxidative emergency reactions.

H2O2 and titanium sulfate generate a yellow titanium peroxide complex with the characteristic absorption at 415 nm.

Reagents and Equipment Required but Not Provided.

Spectrophotometer/microplate reader, desk centrifuge, adjustable pipette, micro glass cuvette/96 well flat-bottom plate, transfer pipette, acetone concentrated sulfuric acid (37% HCl), mortar and ice.

Procedure:

I. Sample Extraction:

1. 1. Bacterial or cell sample: collect bacterial or cell sample to centrifuge, discard the supernatant; suggested 5 million with 1mL of regent I, splitting bacteria and cell with ultrasonication (power 20%, work time 3s, interval 10s, for 30 times); centrifuge at 8000g and 4℃ for 10 min, supernatant is placed on ice for test.
   2. Tissue: take 0.1g tissue add 1 ml regent I, fully grinding on ice. Centrifuge at, 8000g and 4℃for 10 min, supernatant is placed on ice for test.
   3. Serum: according to the proportion of per 100μL of serum (plasma) add 0.9mL regent I, mix well. Centrifuge at 8000g and 4℃ for 10 min, supernatant is placed on ice for test.

II. Determination procedure:

• Preheat spectrophotometer/microplate reader for 30 min, adjust wavelength to 415 nm, set zero with distilled water.
• Incubate Solution Ⅱ, Ⅲ and Ⅳ at 37℃(mammals) or 25℃ (other animals) water bath for more than 10 min minutes.
• Standard working solution: If using a 96-well plate, dilute the 1mmol/mL standard solution to 2μmol/mL standard solution with acetone, and use a trace glass colorimetric method to dilute 1mmol/mL standard solution to 1μmol/mL standard solution
• Add reagents with the following list (reaction in EP tube):

Add Regent Ⅳ to dissolve the precipitate (the step can remove the vegetable pigment with acetone for 3-5 times), and place it at room temperature for 5 min, Transfer 200 μL to a micro glass cuvette or 96-well plate and measure the absorbance at 415 nm. The control tube need only be tested once or twice. Calculate ∆AT  =AT-AC, ∆AS=AS-AC.

III. Calculation (For Microplate reader)

A. 96-well plate

• Cell amount

H2O2(μmol /104 cell) = ∆AT÷(∆AS÷C)×Vs÷(500×Vs÷Ve)=0.004 × ∆AT ÷ ∆AS

• Sample weight

H2O2(μmol/g)= ∆AT÷(∆AS÷C)×V1÷(Vs÷Ve×W)= 2×∆AT ÷ ∆AS ÷ W

• Protein concentration

H2O2(μmol/mg prot)= ∆AT ÷(∆AS÷C)×Vs÷(Cpr×Vs)= 2×∆AT÷ ∆AS ÷ Cpr

• Serum (plasma)volume

H2O2(μmol/mL)= ∆AT  ÷(∆AS÷C)×10=20 × ∆AT÷ ∆AS

500: cell or bacteria amount, 104;

C: concentration of H2O2 standard solution, 2μmol/mL;

Vs: sample volume, 0.25 ml; W: Sample weight, g;

Ve: extraction volume, 1 ml;

Cpr: sample protein concentration, mg/mL;

10: serum dilution multiple. [0.1mL serum (plasma)+0.9mL regent I]÷0.1mL serum(plasma)=10.

B. micro glass cuvette:

Change the concentration of standard C-2μmol/mL in the above formula to C-1μmol/mL for calculation.

Note:

1. As Solution, I is easily volatile, Solution I must be precooled before use. It must be ground on ice when grinding.
2. The solution in this kit is easily volatile. Please bring disposable gloves and masks.
3. If the absorbance value of the sample is greater than 1.1, it is recommended to dilute the sample with Reagent I before performing the measurement.

Experimental examples:

1. Take 0.1 g of heart and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceed according to the determination procedure. After determination with 96 well flat-bottom plate, calculate ∆AT=AT-AC=0.083-0.046=0.039, ∆AS=AS-AC=0.824-0.046=0.778. The content is calculated according to the sample mass.

H2O2(μmol/g) =2×∆AT ÷ ∆AS ÷ W =1 μmol/g.

2. Take 0.1 g of tea and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceed according to the determination procedure. After determination with 96 well flat-bottom plate, calculate ∆AT=AT-AC=0.258-0.003=0.255, ∆AS=AS-AC=0.637-0.003=0.634. The content is calculated according to the sample mass.

H2O2(μmol/g) =2×∆AT ÷ ∆AS ÷ W =4.5 μmol/g.

References：

• Satterfield C N, Bonnell A interference in titanium sulfate method for hydrogen peroxide[J].

Analytical Chemistry, 1955, 27(7): 1174-1175.

• Amin V M, Olson N F. Spectrophotometric determination of hydrogen peroxide in milk[J]. Journal of Dairy Science, 1967, 50(4):461-464.
• Sima Y H, Yao J M, Hou Y S, et al. Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination[J]. Archives of insect biochemistry and physiology, 2011, 77(2): 72-80.

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Technical Specifications:

Limit of Detection  ：0.0027 μmol/mL

Linear Range：0.0195-3 μmol/mL