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过氧化氢酶（Catalase，CAT）又称触酶，是一类以铁卟啉为辅基的结合酶，由四个相同亚单位组成的四聚体酶，共含4分子的亚铁血红素作为辅基，分子量约为24KD，CAT能将细胞代谢产生的毒性物质过氧化氢迅速清除，可与GSH-Px共同保护巯基酶、膜蛋白、过氧化氢解离。该酶的检测对于研究植物代谢强度及抗旱、抗病能力有一定的价值。
检测原理：血清或血浆等样品H₂O₂在240nm处有强烈吸收，过氧化氢酶能分解过H₂O₂，使待测溶液吸光度随反应时间而减少，通过紫外酶标仪测定240nm处吸光度，根据测定吸光度的变化速度即可测出过氧化氢酶的活性。
适用样本：动植物组织；血清；血浆；细胞；细菌
| H1508206 | Component | 100T | Storage |
| H1508206A | H₂O₂基液 | 1 mL×2 | 2-8℃. Store in the dark. |
| H1508206B | CAT Assay Buffer (2.5×) | 250 mL×2 | 2-8℃ |
自备仪器和试剂
操作步骤
1、配制CAT Assay Buffer工作液
按CAT Assay Buffer (2.5×)：蒸馏水=1：1.5的比例稀释，即获得CAT Assay Buffer工作液，4℃预冷待用。
2、配制100mM H₂O₂基液
该试剂盒提供的H₂O₂基液中的H₂O₂浓度约为1M，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，把浓度约为1M的H₂O₂基液用CAT Assay Buffer工作液稀释100倍，使H₂O₂浓度约为10mM；分光光度计测定A240（一般情况下新配制的10mM H₂O₂基液A240在0.45左右，经过3个月以后A240在0.42左右）， H₂O₂浓度 (mM)=22.94×A240。从而计算出该试剂盒提供的H₂O₂基液中H₂O₂的实际浓度，然后根据实际的H₂O₂浓度，配制100mM H₂O₂基液。
3、样品准备
3.1 植物、动物样品
取正常或逆境下的新鲜植物组织（或动物组织），清洗干净，擦干，切碎，迅速称取，按照组织质量(g)：CAT Assay buffer工作液体积(mL)为1:5-10的比例（建议称取约 0.1g组织，加入1mL CAT Assay buffer工作液），进行冰浴匀浆。8000g 4℃离心10分钟，取上清，置冰上待测。
3.2 血浆、血清和尿液样品
血浆、血清按照常规方法制备，用生理盐水10倍稀释后，可以直接用于本试剂盒的测定，尿液通常也可以直接用于测定，置冰上待测。
3.3 细菌或培养细胞样品
收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量(10⁴个)：CAT Assay buffer工作液体积(mL)为500-1000：1的比例（建议500万细菌或细胞加入1mL CAT Assay buffer工作液），超声波破碎细菌或细胞（功率200w，超声3秒，间隔10秒，重复30次）；8000g 4℃离心10分钟，取上清，置冰上待测。
4、CAT加样
取96孔UV板，按照下表设置空白孔、测定孔，溶液应按照顺序依次加入，并注意避免产生气泡；如果样品中的酶活性过高，可以减少样品用量或适当稀释后再进行测定，样品的测定最好设置平行复测孔。
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5、CAT测定
加入100mM H₂O₂基液24μl，每加完一孔立即计时，亦可用排枪同时加样，以避免误差；蒸馏水调零，酶标仪测定240nm处各孔吸光度，每隔1min读数1次，共测4次，待全部测定完毕后，计算酶活力。
6、结果计算
6.1 植物、动物组织中CAT活力的计算
(1) 按样本质量计算
单位定义：每g组织在25℃条件下，240nm处吸光度值每分钟下降0.1定义为一个CAT酶活力单位。
CAT[U/(g·min)]=(ΔA240×VT×N)/(0.1×VS×t×W)
(2) 按蛋白浓度计算
单位定义：每mg组织蛋白在25℃条件下，240nm处吸光度值每分钟下降0.1定义为一个CAT酶活力单位。
CAT[U/(mg·min)]=(ΔA240×VT×N)/(0.1×VS×t×Cpr)
6.2 血清、血浆、尿液中CAT活力的计算
单位定义：每ml液体样本在25℃条件下，240nm处吸光度值每分钟下降0.1定义为一个CAT酶活力单位。
CAT[U/(ml·min)]=(ΔA240×N)/(0.1×VS×t)
6.3 细菌/细胞中CAT活力的计算
单位定义：每1万个细菌或细胞样本在25℃条件下，240nm处吸光度值每分钟下降0.1定义为一个CAT酶活力单位。
CAT[U/(10⁴cell·min)]=(ΔA240×VT×N)/(0.1×VS×t×n)
参数说明：
ΔA240=A空白-(A测定Ⅰ+A测定Ⅱ)/2
A空白：空白的吸光度
A测定：待测样品最后比较稳定的吸光度
VT：提取酶液总体积(ml)
N：待测样品检测前的稀释倍数
VS：测定时所用样品体积(ml)
t：加H₂O₂基液到最后一次读数的反应时间(min)
W：样品新鲜质量(g)
n：细胞或细菌数量，500~1000，单位为万
0.1：A240下降0.1时的一个酶活力单位
注意事项
1、待测样品中不应含有CAT抑制剂，同时需避免反复冻融。
2、400nm以下吸光度值的测定应选用石英比色杯或UV酶标板。
3、CAT Assay Buffer如出现沉淀或絮状物，可用50℃左右温水助溶，仍有絮状物应弃用。
4、完整的红细胞以及未稀释的溶血液中的过氧化氢酶置于4℃ 1周仍然很稳定，稀释后的溶血液中CAT容易失活。
5、尽量避免冰冻样品造成溶血，否则过氧化氢酶活性会下降10%～15%。
6、血清样品3天内室温下活性下降64.7%，4℃下下降10.5%，-20℃保存30天活性仅下降3.5%，因此待测样品均应-20℃或-70℃保存。
7、如果反应液有大量气泡产生，将样本用蒸馏水稀释后再进行测定。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
9、试剂开封后请尽快使用，以防影响后续实验效果。
10、该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
Catalase (CAT), also known as hematase, is a conjugated enzyme that uses iron porphyrin as its prosthetic group. It is a tetrameric enzyme composed of four identical subunits, containing four molecules of heme as cofactors, with a molecular weight of approximately 24 kDa. CAT rapidly eliminates hydrogen peroxide, a toxic substance produced by cell metabolism, and works together with GSH‑Px to protect sulfhydryl enzymes, membrane proteins, and to dissociate hydrogen peroxide. The detection of this enzyme holds certain value for studying plant metabolic intensity as well as drought and disease resistance.
Detection Principle
Hydrogen peroxide (H₂O₂) in samples such as serum or plasma has strong absorption at 240 nm. Catalase decomposes H₂O₂, causing the absorbance of the test solution to decrease over time. The activity of catalase can be determined by measuring the rate of change in absorbance at 240 nm using a UV microplate reader.
Applicable Samples: Animal and plant tissues; Serum; Plasma; Cells; Bacteria
| H1508206 | Component | 100T | Storage |
| H1508206A | H₂O₂ Stock Solution | 1 mL×2 | 2-8℃. Store in the dark. |
| H1508206B | CAT Assay Buffer (2.5×) | 250 mL×2 | 2-8℃ |
Reagents, consumables and Equipments not provided
Full‑wavelength microplate reader, Spectrophotometer (capable of measuring absorbance at 240 nm)
96‑well UV plate
Mortar or homogenizer, Centrifuge tubes, Centrifuge, Water bath or incubator
Distilled water, Physiological saline
Procedure
1. Preparation of CAT Assay Buffer Working Solution
Dilute CAT Assay Buffer (2.5×) with distilled water at a ratio of 1:1.5 to obtain the CAT Assay Buffer Working Solution. Pre‑cool at 4°C for later use.
2. Preparation of 100 mM H₂O₂ Stock Solution
The H₂O₂ stock solution provided in this kit has an approximate H₂O₂ concentration of 1 M. Since hydrogen peroxide is not very stable, its actual concentration must be determined before use. Dilute the approximately 1 M H₂O₂ stock solution 100‑fold with CAT Assay Buffer Working Solution to obtain an approximately 10 mM H₂O₂ solution. Measure its absorbance at 240 nm (A<sub>240</sub>) using a spectrophotometer (typically, a freshly prepared 10 mM H₂O₂ solution has an A<sub>240</sub> around 0.45, which may drop to around 0.42 after 3 months). Calculate the H₂O₂ concentration (mM) using the formula: H₂O₂ concentration (mM) = 22.94 × A<sub>240</sub>. This gives the actual H₂O₂ concentration in the stock solution provided. Based on this actual concentration, prepare a 100 mM H₂O₂ stock solution accordingly.
3. Sample Preparation
3.1 Plant and Animal Samples
Take fresh plant or animal tissue (normal or under stress), wash clean, dry, cut into small pieces, and quickly weigh. Homogenize on ice using a tissue mass (g) to CAT Assay Buffer Working Solution volume (mL) ratio of 1:5‑10 (recommended: weigh about 0.1 g tissue and add 1 mL CAT Assay Buffer Working Solution). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for testing.
3.2 Plasma, Serum, and Urine Samples
Prepare plasma and serum using standard methods. Dilute 10‑fold with physiological saline, then they can be directly used for assay with this kit. Urine can usually be tested directly. Keep samples on ice.
3.3 Bacterial or Cultured Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Based on a ratio of bacterial/cell count (10⁴) to CAT Assay Buffer Working Solution volume (mL) of 500‑1000:1 (recommended: add 1 mL CAT Assay Buffer Working Solution per 5 million bacteria or cells), disrupt the bacteria or cells by sonication (power 200 W, pulse 3 seconds, interval 10 seconds, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for testing.
4. CAT Sample Loading
Use a 96‑well UV plate. Set up blank and test wells according to the table below. Add solutions in the specified order, avoiding bubble formation. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is best to set up replicate wells for each sample.
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5. CAT Assay
Add 24 µL of 100 mM H₂O₂ Stock Solution. Start timing immediately after adding to each well (a multichannel pipette can be used for simultaneous addition to minimize error). Zero the instrument with distilled water. Measure the absorbance at 240 nm for each well using the microplate reader, taking readings every 1 minute for a total of 4 readings. After all measurements are completed, calculate the enzyme activity.
6. Calculation of Results
6.1 Calculation of CAT Activity in Plant/Animal Tissues
(1) Based on sample mass
Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per gram of tissue at 25°C.
CAT [U/(g·min)] = (ΔA240 × VT × N) / (0.1 × VS × t × W)
(2) Based on protein concentration
Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per milligram of tissue protein at 25°C.
CAT [U/(mg·min)] = (ΔA240 × VT × N) / (0.1 × VS × t × Cpr)
6.2 Calculation of CAT Activity in Serum, Plasma, and Urine
Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per milliliter of liquid sample at 25°C.
CAT [U/(mL·min)] = (ΔA240 × N) / (0.1 × VS × t)
6.3 Calculation of CAT Activity in Bacteria/Cells
Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per 10⁴ bacterial or cell sample at 25°C.
CAT [U/(10⁴ cell·min)] = (ΔA240 × VT × N) / (0.1 × VS × t × n)
Parameter Description:
ΔA240 = ABlank – (ATest I + ATest II) / 2
ABlank: Absorbance of the blank
A<sub>Test</sub>: The final, relatively stable absorbance of the test sample
V<sub>T</sub>: Total volume of enzyme extraction solution (mL)
N: Dilution factor of the test sample before measurement
VS: Volume of sample used in the assay (mL)
t: Reaction time from addition of H₂O₂ stock solution to the last reading (min)
W: Fresh weight of the sample (g)
n: Number of cells or bacteria, in units of 10⁴ (500~1000)
0.1: One enzyme activity unit corresponds to a decrease in A<sub>240</sub> of 0.1
Notes
Test samples should not contain CAT inhibitors, and repeated freeze‑thaw cycles should be avoided.
For absorbance measurements below 400 nm, quartz cuvettes or UV‑compatible microplates should be used.
If precipitates or flocculants appear in the CAT Assay Buffer, warming in approximately 50°C water may help dissolve them. If flocculants persist, discard the buffer.
Intact red blood cells and undiluted hemolysate maintain stable catalase activity for up to 1 week at 4°C. CAT in diluted hemolysate is prone to inactivation.
Avoid hemolysis caused by freezing samples, as it can reduce catalase activity by 10‑15%.
Serum samples lose 64.7% activity at room temperature within 3 days, 10.5% at 4°C, but only 3.5% after 30 days at ‑20°C. Therefore, test samples should be stored at ‑20°C or ‑70°C.
If a large number of bubbles form in the reaction mixture, dilute the sample with distilled water before measurement.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0332376 |
分析证书 | H1508206 |
危险品化学品经营许可证（带存储）