过氧化氢酶是一种以铁卟啉为辅基的关键抗氧化酶，广泛存在于动物、植物、微生物和培养细胞中。其主要功能是催化过氧化氢分解为水和氧气。H₂O₂是细胞有氧代谢产生的活性氧类物质，能直接或间接氧化细胞内核酸、蛋白质、脂类等生物大分子，导致细胞损伤、加速衰老和解体。过氧化氢酶通过清除体内的H₂O₂，使细胞免遭其毒害，是生物防御体系的关键酶之一。
本试剂盒的原理为：过氧化氢酶催化底物H₂O₂分解为水和氧气，消耗体系中定量的H₂O₂。剩余的H₂O₂在4-氨基安替吡啉和酚存在时，经过氧化物酶催化，反应生成红色醌类化合物，在520 nm处有吸收峰，其颜色深浅与剩余H₂O₂浓度成正比。因此，酶活性越高，消耗底物越多，吸光值越低。用未加酶的对照孔吸光值减去样本孔吸光值所得差值（即吸光度减少值）直接反映被分解的H₂O₂量，该减少值与酶活性呈正比。根据吸光度减少值与标准品活性之间的关系进行四参数拟合建立标准曲线，从而实现样本酶活性的定量。
产品组分
C1511511 | 组件 | 物理外观 | 100 T | 储存 | 单次测试使用量 |
C1511511A | 裂解液 | 液体 | 30 mL | -20℃ | 按需 |
C1511511B | 检测缓冲液 | 液体 | 30 mL | -20℃ | 按需 |
C1511511C | 过氧化氢（1M） | 液体 | 2 mL | -20℃.避光 | 按需 |
C1511511D | 辣根过氧化物酶 | 液体 | 200 μL | -20℃.避光 | 2 μL |
C1511511E | 显色试剂 | 液体 | 700 μL | -20℃.避光 | 7 μL |
C1511511F | 过氧化氢酶标准品（200 U/mL） | 液体 | 500 μL | -20℃.避光 | 5 μL |
使用方法
1. 样品准备
（1）血液样本。处理好的血清血浆样本直接检测或者用检测缓冲液稀释后进行检测。
（2）细胞样本。收集细胞，PBS洗涤一次，按照每100万细胞加入100-200 μL裂解液的比例加入裂解液，冰浴裂解5-10分钟或超声匀浆。4ºC约12,000×g离心5分钟，取上清用于后续检测。
（3）组织样本。按照每10 mg组织加入100 μL 裂解液的比例，冰浴等低温条件下进行匀浆。4ºC约12,000×g离心3-5分钟，取上清用于后续检测。
以上所有操作均需在4ºC或冰上操作。制备好的细胞或组织样品如果不能立即检测，可以-20ºC或-80ºC冻存。
2、过氧化氢酶标准曲线的设置
取7支EP管，第一支EP管中加入5 µL过氧化氢酶标准品（200 U/mL）和195 µL的检测缓冲液或裂解液（对于不需要裂解液处理的样本使用检测缓冲液，对于需要裂解液处理的样本使用裂解液），将过氧化氢酶稀释为5 U/mL。其他每管中加入100 μL检测缓冲液或裂解液。第一只EP管中吸取100 μL第2支EP管，按此步骤往后依次稀释，最后一支EP管为100 μL检测缓冲液或裂解液。此时，各个EP管中过氧化氢酶的浓度依次为5、2.5、1.25、0.625、0.313、0.156、0 U/mL。
3、过氧化氢工作液的配制
由于过氧化氢不是非常稳定，使用前需自行测定过氧化氢的实际浓度以进行校准。把浓度约为1 M的过氧化氢用水稀释100倍，使过氧化氢的浓度约为10 mM，测定A240。A240的测定可采用如下的任一方法：普通紫外分光光度计法、微量紫外分光光度计法、96孔紫外酶标仪法，其中用比色皿检测的过氧化氢浓度最接近实际浓度。以光程为1 cm的比色皿校准为例，用下列公式计算计算过氧化氢的浓度：过氧化氢浓度(mM)=22.94 ×A240。得到结果后再乘以稀释倍数100即为过氧化氢校准后的浓度。
以校准后的过氧化氢浓度为准，将过氧化氢稀释为2 mM的过氧化氢工作液。检测样本时，标准孔和样品孔各加入25 µL过氧化氢工作液，按需配置，现配现用，配制完成后尽快使用。
4. 显色液的配制
按照每个反应50 µL的体积配制适量的显色液。均匀混合41 µL检测缓冲液、2 µL 辣根过氧化物酶和7 µL显色试剂，即可配制成50 µL显色液。根据待检测样品(包括标准品)的数量，配制适量的反应工作液，建议尽量现配现用。具体配制方法参考下表：
样本数 | 1 | 10 | 20 | 50 |
检测缓冲液 | 41 µL | 410 µL | 820 µL | 2050 µL |
辣根过氧化物酶 | 2 µL | 20 µL | 40 µL | 100 µL |
显色试剂 | 7 µL | 70 µL | 140 µL | 350 µL |
总体积 | 50 µL | 500 µL | 1000 µL | 2500 µL |
5. 样品测定
（1）取25 μL标准品和样品加入透明96孔板中，同时设置仅含裂解液或检测缓冲液的孔为空白对照。再向标准品孔和样品孔中加入25 μL的底物过氧化氢工作液，可以设置一个不含过氧化氢工作液的孔作为背景对照孔，即25 μL的过氧化氢工作液用25 μL的检测缓冲液替代。室温避光反应10 min。
（2）向标准品孔和样品孔中加入显色液50 µL，混匀，室温避光反应10分钟。
（3）酶标仪读取OD520。
6. 结果计算
标准曲线中不含过氧化氢酶的空白对照孔的值为A1，标准孔的值为A2，则各个标准孔的OD减少值为A1-A2。以过氧化氢酶的酶活为横坐标，OD减少值为纵坐标，使用四参数拟合绘制标准曲线。
标准曲线中不含过氧化氢酶的空白对照孔的值减去样品OD值为样品OD减少值。将样品OD减少值带入标准曲线中，即可得到样品中过氧化氢酶的酶活。
7. 结果展示
标准曲线
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注意事项
1. 为减少稀释液产生的误差，样品和标准品的稀释液应该根据样品的种类来定。当样品为裂解液制备的细胞或组织的裂解样品时，应使用裂解液稀释，当样品为血液等其它样品时，宜使用检测缓冲液稀释。
2. 过氧化氢的浓度需根参考说明书中的方法进行测定，后续按照测定的实际浓度进行实验。
3. 过氧化氢酶标准品使用前后至于冰上保存，请勿长期室温放置。
4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
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
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.
ZJ26F0737920
危险品化学品经营许可证（带存储）