Catalase is a key antioxidant enzyme with iron porphyrin as its prosthetic group, widely present in animals, plants, microorganisms, and cultured cells. Its main function is to catalyze the decomposition of hydrogen peroxide into water and oxygen. H₂O₂ is a reactive oxygen species produced by cellular aerobic metabolism, which can directly or indirectly oxidize nucleic acids, proteins, lipids, and other biological macromolecules, leading to cell damage, accelerated aging, and disintegration. By scavenging H₂O₂, catalase protects cells from its toxicity and is one of the key enzymes in the biological defense system.
The principle of this kit is as follows: Catalase catalyzes the decomposition of the substrate H₂O₂ into water and oxygen, consuming a defined amount of H₂O₂ in the system. In the presence of 4-aminoantipyrine and phenol, the remaining H₂O₂ is catalyzed by peroxidase to form a red quinone compound, which has an absorption peak at 520 nm. The color intensity is directly proportional to the remaining H₂O₂ concentration. Therefore, higher enzyme activity results in greater substrate consumption and lower absorbance. The difference obtained by subtracting the absorbance of the sample well from that of the control well without enzyme (i.e., the absorbance decrease value) directly reflects the amount of H₂O₂ decomposed, and this decrease is proportional to the enzyme activity. A standard curve is established by four-parameter logistic fit based on the relationship between the absorbance decrease and the activity of the standard, thereby allowing quantification of enzyme activity in the sample..
Components
C1511511 | Components | Appearance | 100 T | Storage | Quantity Per Test |
C1511511A | Lysis Buffer | Liquid | 30 mL | -20°C | As needed |
C1511511B | Assay Buffer | Liquid | 30 mL | -20°C | As needed |
C1511511C | Hydrogen peroxide (1 M) | Liquid | 2 mL | -20°C. Store in the dark | As needed |
C1511511D | Horseradish Peroxidase | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
C1511511E | Chromogenic Reagent | Liquid | 700 μL | -20°C. Store in the dark | 7 μL |
C1511511F | Catalase standard (200 U/mL) | Liquid | 500 μL | -20°C. Store in the dark | 5 μL |
Usage Protocol
1. Sample Preparation
1) Blood samples. For properly prepared serum or plasma samples, they can be directly assayed or diluted with assay buffer before measurement.
2) Cell samples. Collect cells, wash once with PBS. Add lysis buffer at a ratio of 100–200 μL per 1 × 10⁶ cells, and lyse on ice for 5–10 minutes or by sonication homogenization. Centrifuge at approximately 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for subsequent analysis.
3) Tissue samples. Add lysis buffer at a ratio of 100 μL per 10 mg of tissue, and homogenize under low‑temperature conditions such as on ice. Centrifuge at approximately 12,000 × g for 3–5 minutes at 4 °C, and collect the supernatant for subsequent analysis.
All of the above operations should be performed at 4 °C or on ice. If the prepared cell or tissue samples cannot be analyzed immediately, they may be stored at –20 °C or –80 °C.
2. Preparation of Catalase Standard Curve
Take seven EP tubes. To the first EP tube, add 5 µL of catalase standard (200 U/mL) and 195 µL of assay buffer or lysis buffer (use assay buffer for samples that do not require lysis treatment; use lysis buffer for samples that require lysis treatment) to dilute the catalase to 5 U/mL. Add 100 μL of assay buffer or lysis buffer to each of the remaining tubes. Transfer 100 μL from the first tube to the second tube, and continue this serial dilution through the subsequent tubes. The last tube contains 100 μL of assay buffer or lysis buffer. The resulting catalase concentrations in each tube are 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0 U/mL, respectively.
3. Preparation of Hydrogen Peroxide Working Solution
Since hydrogen peroxide is not very stable, its actual concentration must be determined by the user before use for calibration. Dilute the approximately 1 M hydrogen peroxide solution 100‑fold with water to obtain a concentration of approximately 10 mM, and measure A₂₄₀. The A₂₄₀ measurement can be performed by any of the following methods: ordinary UV spectrophotometer, micro‑volume UV spectrophotometer, or 96‑well UV microplate reader. Among these, the hydrogen peroxide concentration measured using a cuvette is closest to the actual concentration. Taking calibration using a cuvette with a 1 cm pathlength as an example, calculate the hydrogen peroxide concentration using the following formula: Hydrogen peroxide concentration (mM) = 22.94 × A₂₄₀. Multiply the result by the dilution factor of 100 to obtain the calibrated concentration of hydrogen peroxide.
Based on the calibrated hydrogen peroxide concentration, dilute the hydrogen peroxide to prepare a 2 mM hydrogen peroxide working solution. For sample detection, add 25 μL of the hydrogen peroxide working solution to each standard well and sample well. Prepare the amount needed, and use it immediately after preparation; it should be used as soon as possible.
4. Preparation of Chromogenic Solution
Prepare an appropriate volume of Chromogenic Solution based on a volume of 50 µL per reaction. Mix 41 µL of Assay Buffer, 2 µL of Horseradish Peroxidase, and 7 µL of Chromogenic Reagent to prepare 50 µL of Chromogenic Solution. Prepare the appropriate amount of Reaction Working Solution according to the number of samples to be tested (including standards). It is recommended to prepare the solution fresh before use. Refer to the table below for specific preparation methods:
Number of samples | 1 | 10 | 20 | 50 |
Assay Buffer | 41 µL | 410 µL | 820 µL | 2050 µL |
Horseradish Peroxidase | 2 µL | 20 µL | 40 µL | 100 µL |
Chromogenic Reagent | 7 µL | 70 µL | 140 µL | 350 µL |
Total volume | 50 µL | 500 µL | 1000 µL | 2500 µL |
5. Sample Determination
1) Add 25 μL of standard and sample to a clear 96‑well plate, and set up wells containing only lysis buffer or assay buffer as blank controls. Then add 25 μL of substrate hydrogen peroxide working solution to the standard wells and sample wells. A well without hydrogen peroxide working solution can be set as a background control well, i.e., replace the 25 μL of hydrogen peroxide working solution with 25 μL of assay buffer. Incubate at room temperature in the dark for 10 min.
2) Add 50 µL of Chromogenic Solution to the standard wells and sample wells, mix well, and incubate at room temperature in the dark for 10 minutes.
3) Read the absorbance at 520 nm (OD520) using a microplate reader..
6. Result Calculation
The absorbance value of the blank control well (without catalase) in the standard curve is designated as A1, and that of the standard wells as A2. The OD decrease value for each standard well is calculated as A1 – A2. A standard curve is then constructed by plotting catalase activity on the x‑axis against the OD decrease value on the y‑axis, using a four‑parameter logistic fit.
The OD decrease value for a sample is obtained by subtracting the sample OD value from that of the blank control well (without catalase) in the standard curve. Substituting this sample OD decrease value into the standard curve yields the catalase activity in the sample.
7. Results Presentation
Standard curve
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Precautions
1. To reduce errors caused by the diluent, the diluent for samples and standards should be selected according to the sample type. When the sample is a cell or tissue lysate prepared with lysis buffer, lysis buffer should be used for dilution; for other samples such as blood, assay buffer should be used.
2. The concentration of hydrogen peroxide should be determined according to the method described in the manual, and subsequent experiments should be performed based on the actual measured concentration.
3. The catalase standard should be kept on ice before and after use; do not leave it at room temperature for extended periods.
4. For your safety and health, please wear a lab coat and disposable gloves during operation.
