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。
检测原理：血清或血浆等样本在最佳酶反应条件下，反应后剩余的H₂O₂与钼酸铵形成稳定的黄色复合物，其黄色深浅与酶活性成反比，通过分光光度计测定405nm处吸光度。该酶的检测对于研究自由基代谢平衡、抗衰老和肿瘤发病机制具有一定的价值。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 50T | 100T | 储存 |
| C1505482NT | 过氧化氢酶 (CAT) 活性检测试剂盒 (钼酸铵, 比色法) (常温组分) | C1505482B | CAT Assay Buffer | 液体 | 50mL | 100mL | 室温 |
| | C1505482C | 钼酸铵试剂 | 固体 | 5g | 10g | 室温 |
| C1505482WT | 过氧化氢酶 (CAT) 活性检测试剂盒 (钼酸铵, 比色法) (2-8℃组分) | C1505482A | H₂O₂基液 | 液体 | 5ml | 10mL | 2-8℃. 避光 |
注：发货时各组分依据储存温度（室温/ 2-8℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。
自备仪器和试剂
1、蒸馏水、生理盐水
2、研钵或匀浆器、离心管、离心机、水浴锅或恒温箱、分光光度计、比色杯
实验流程
1、配制65mM H₂O₂基液
本试剂盒提供的H₂O₂基液中的H₂O₂浓度约为1M，由于H₂O₂不是非常稳定，使用前需自行测定H₂O₂的实际浓度，把浓度约为1M的H₂O₂基液用CAT Assay Buffer稀释100倍，使H₂O₂基液中的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0.4~0.45左右，经过3个月以后A240;在0.35~0.4左右)，H₂O₂浓度(mM)=22.94×A240;，进而计算出本试剂盒提供的H₂O₂基液中的H₂O₂的实际浓度，然后再根据实际的H₂O₂浓度，配制65mM H₂O₂基液。
2、配置MO显色液
称取0.4g钼酸铵试剂加入到10ml蒸馏水中，充分溶解即成，MO显色液易出现乳白色样物质，应弃用，尽量现用现配，该试剂盒提供的钼酸铵试剂为过量。
3、样品准备
3.1 细胞或组织样品
取恰当细胞或组织进行裂解，可以采用RIPA裂解液，如有必要可进行适当匀浆，低速离心取上清，-20℃冻存，用于CAT的检测。
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测定，不能及时检测可-20℃冻存。
3.3 全血样品
收集适量的全血至一抗凝管内，颠倒混匀，取全血冻融一次，用CAT Assay Buffer 1000倍后进行CAT检测。
3.4 血液中的红细胞裂解液
用抗凝管收集血液，颠倒混匀，取至少500μl全血4℃ 3000g离心5min，弃上清，沉淀用预冷的生理盐水洗涤3次，用约5倍细胞体积的预冷的去离子水，例如Milli-Q纯水，重悬细胞沉淀，冰浴10min；使用前用CAT Assay Buffer稀释400倍后进行CAT检测。
3.5 植物样品
准确称取植物材料（果肉或者去叶脉的叶片）0.5g，剪碎，置于4℃预冷的研钵或匀浆器中，加入预冷提取液1ml，低温研磨至匀浆后转移至离心管，用3ml提取液冲洗研钵或匀浆器并转入离心管，加提取液至总体积为5ml；4℃ 10000r/min离心20min，上清液为酶提取液，上清液可用于CAT的检测。提取液配方：无水磷酸氢二钠3.3g+二水磷酸二氢钠0.13g，补水至200ml，完全溶解即为提取液，4℃保存备用。
注意：如果CAT酶活性较低，应相应减少提取液的总体积，以便提高CAT酶的浓度。
3.6 高活性样品
如果样品中含有较高活性的CAT，可以使用CAT Assay Buffer稀释。
3.7 （选做）样品准备完毕后可以用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内的CAT含量。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
4、CAT加样
按照下表设置空白管、自身对照管、测定管，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，样品的测定最好能设置平行管。
加入物 (ml)
| 空白管
| 自身对照管
| 测定管 |
CAT Assay Buffer
| 0.1 | —
| —
|
待测样品
| —
| —
| 0.1
|
65mM H₂O₂基液 (37℃预温5min)
| 1.0 | 1.0
| 1.0
|
立即混匀，置于37℃水浴，准确孵育60s。
5、CAT测定
分光光度计测定405nm处吸光度，读取各管吸光度（分别记为A空白;、A自身对照;、A测定;）。如果没有分光光度计，亦可用酶标仪测定，但如果有条件，尽量采用分光光度计测定。
6、结果计算
CAT活性单位的定义：在37℃ 1min催化水解1μmol H₂O₂量为一个CAT酶活力单位，根据酶活性定义，计算出样品中的CAT活性。
6.1 血清、血浆、尿液中CAT活力计算公式：
血清样品CAT活力 (U/ml)=｛(A自身对照;-A测定;)/A空白;｝×650×N
6.2 组织、细胞中CAT活力计算公式：
组织样品CAT活力 (U/mg)=｛(A自身对照;-A测定;)/A空白;｝×650/待测样品血红蛋白浓度(mg/ml)
6.3 植物组织中CAT活力计算公式：
植物样品CAT活力 (U/g)=｛(A自身对照;-A测定;)/A空白;｝×650×N×V/m
参数说明：
A自身对照;：自身对照的吸光度；
A测定;：待测样品的吸光度；
A空白;：空白的吸光度；
N：待测样品检测前的稀释倍数；
V：提取液总体积(ml)；
m：植物样品的质量(g)=0.5。
注意事项
1、该试剂盒亦可用酶标仪进行检测，但测定的样本数相应增多，如果有条件，尽量采用分光光度计检测。
2、待测样品中不应含有CAT抑制剂，同时需避免反复冻融。
3、CAT Assay Buffer如果出现浑浊或絮状物，应弃用。
4、MO显色液配置后可4℃稳定两周，如果出现乳白色物质应弃用，最好现用现配。
5、对于植物样品研磨处理应迅速，以免CAT酶活下降，同时尽量置于冰浴状态处理；另外本法不推荐用于植物样本CAT的检测，最好采用C661945 过氧化氢酶活性检测试剂盒（紫外吸收法）。
6、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周仍然较稳定，稀释后的溶血液中CAT容易失活。
7、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%。
8、血清样品室温下3天内活性下降64.7%，4℃条件下下降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。
9、为了您的安全和健康，请穿实验服并戴一次性手套操作。
10、试剂开封后请尽快使用，以防影响后续实验效果。
Catalase (CAT), also known as hydroperoxidase, is a conjugated enzyme with an iron porphyrin prosthetic group. It is a tetrameric enzyme composed of four identical subunits, containing four molecules of heme as prosthetic groups, with a molecular weight of approximately 24 kD. CAT rapidly scavenges hydrogen peroxide, a toxic substance produced by cellular metabolism, and works together with GSH-Px to protect sulfhydryl enzymes, membrane proteins, and facilitate hydrogen peroxide decomposition.
Assay Principle
Under optimal enzymatic reaction conditions, the residual H₂O₂ in samples like serum or plasma reacts with ammonium molybdate to form a stable yellow complex. The intensity of the yellow color is inversely proportional to the enzyme activity. The absorbance at 405 nm is measured using a spectrophotometer. The detection of this enzyme holds value for researching the balance of free radical metabolism, anti-aging mechanisms, and the pathogenesis of tumors. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
| Storage Unit No. | Storage and Transport Unit Name
| Component No. | Component Name | Appearance | 50T | 100T | Storage |
| C1505482NT | Catalase (CAT) Activity Assay Kit (AHM, Colorimetric Method) (RT Components) | C1505482B | CAT Assay Buffer | Liquid | 50mL | 100mL | Room temperature |
| | C1505482C | Ammonium Molybdate Reagent | Solid | 5g | 10g | Room temperature |
| C1505482WT | Catalase (CAT) Activity Assay Kit (AHM, Colorimetric Method) (2-8℃ Components) | C1505482A | H₂O₂ Stock Solution | Liquid | 5ml | 10mL | 2-8℃.Store in the dark. |
Note: Each component is individually packaged according to its required storage temperature (Room temperature / 2‑8°C) before shipment. Differentiated temperature‑controlled transportation is adopted to ensure product performance. Orders containing items with different storage‑temperature requirements may be delivered in separate shipments, subject to the goods actually received. After receipt, store each component separately at its specified storage temperature. The shelf‑life is 12 months from the date of manufacture.
Required Materials Not Provided
1. Distilled water, Physiological saline
2. Mortar and pestle or homogenizer, Centrifuge tubes, Centrifuge, Water bath or incubator, Spectrophotometer, Cuvettes
Experimental Procedure
1. Preparation of 65 mM H₂O₂ Substrate Solution
The provided H₂O₂ Stock Solution is approximately 1 M. As H₂O₂ is not very stable, determine its actual concentration before use.
Dilute the ~1 M H₂O₂ Stock Solution 100-fold with CAT Assay Buffer to achieve ~10 mM.
Measure the absorbance at 240 nm (A240) using a spectrophotometer.
Calculate the H₂O₂ concentration using the formula: H₂O₂ concentration (mM) = A240× 22.94.
Using this calculated actual concentration, prepare the 65 mM H₂O₂ Substrate Solution by appropriate dilution with CAT Assay Buffer.
2. Preparation of MO Chromogenic Solution
Weigh 0.4 g of Ammonium Molybdate Reagent and add it to 10 mL of distilled water. Dissolve completely.
Note: The MO Chromogenic Solution may develop a milky-white precipitate; if so, discard it. Prepare fresh before use. The provided Ammonium Molybdate Reagent is supplied in excess.
3. Sample Preparation
3.1 Cell or Tissue Samples
Lyse an appropriate amount of cells or tissue using RIPA Lysis Buffer. Homogenize if necessary.
Centrifuge at low speed and collect the supernatant.
Store at -20°C for CAT detection.
3.2 Plasma, Serum, and Urine Samples
Prepare plasma and serum by conventional methods. Dilute 10-fold with physiological saline before direct assay.
Urine can usually be assayed directly.
Store at -20°C if not assayed immediately.
3.3 Whole Blood Samples
Collect an appropriate volume of whole blood into an anticoagulant tube and mix by inverting.
Freeze-thaw the whole blood once.
Dilute 1000-fold with CAT Assay Buffer before CAT assay.
3.4 Erythrocyte Lysate from Blood
Collect blood in an anticoagulant tube and mix.
Take at least 500 µL whole blood, centrifuge at 3000 g for 5 min at 4°C, discard supernatant.
Wash the pellet 3 times with ice-cold physiological saline.
Resuspend the cell pellet in approximately 5 volumes of ice-cold deionized water (e.g., Milli-Q water).
Incubate on ice for 10 minutes.
Dilute 400-fold with CAT Assay Buffer before CAT assay.
3.5 Plant Samples
Precisely weigh 0.5 g of plant material (pulp or leaves without veins), mince, and place in a pre-chilled (4°C) mortar or homogenizer.
Add 1 mL of pre-chilled Extraction Buffer and grind at low temperature to homogenate.
Transfer to a centrifuge tube, rinse the mortar/homogenizer with 3 mL Extraction Buffer, and combine into the tube.
Adjust the final volume to 5 mL with Extraction Buffer.
Centrifuge at 10,000 rpm/min for 20 min at 4°C. The supernatant is the enzyme extract for CAT assay.
Extraction Buffer Recipe: Dissolve 3.3 g anhydrous Disodium Hydrogen Phosphate and 0.13 g Sodium Dihydrogen Phosphate Dihydrate in water, bring final volume to 200 mL. Store at 4°C.
Note: If CAT activity is low, reduce the total volume of Extraction Buffer to increase the enzyme concentration.
3.6 High-Activity Samples
3.7 (Optional) Protein Concentration Assay
4. CAT Assay Setup
Set up Blank, Self-Control, and Test tubes according to the table below.
Add reagents in the specified order, avoiding bubbles.
For high enzyme activity, reduce sample volume or dilute before assay. Duplicate tubes are recommended.
Reagent (mL)
| Blank Tube
| Self-Control Tube
| Test Tube
|
CAT Assay Buffer
| 0.1 | —
| —
|
Sample
| —
| —
| 0.1
|
65 mM H₂O₂ Substrate Solution (pre-warmed 5min, 37°C)
| 1.0 | 1.0
| 1.0
|
Mix immediately and incubate at 37°C for EXACTLY 60 seconds.
Sample
| —
| 0.1
| —
|
MO Chromogenic Solution
| 1.0
| 1.0
| 1.0 |
5. CAT Measurement
Measure the absorbance at 405 nm using a spectrophotometer.
Record the absorbance for each tube (ABlank, ASelf-Control, ATest).
Note: A microplate reader can be used if a spectrophotometer is unavailable, but a spectrophotometer is preferred.
6. Calculation of Results
Definition of CAT Activity Unit: One unit of CAT enzyme activity is defined as the amount of enzyme that catalyzes the decomposition of 1 μmol of H₂O₂ per minute at 37°C.
6.1 CAT Activity in Serum, Plasma, Urine:
CAT Activity (U/mL) = { (ASelf-Control - ATest) / ABlank } × 650 × N
6.2 CAT Activity in Tissues, Cells (based on protein):
CAT Activity (U/mg prot) = { (ASelf-Control - ATest) / ABlank } × 650 / Cpr
(Where Cpr is the sample protein concentration in mg/mL)
6.3 CAT Activity in Plant Tissues:
CAT Activity (U/g weight) = { (ASelf-Control - ATest) / ABlank} × 650 × N × V / m
Parameter Definitions:
ASelf-Control: Absorbance of Self-Control tube
ATest: Absorbance of Test tube
ABlank: Absorbance of Blank tube
N: Dilution factor of the test sample before assay
V: Total volume of extraction buffer (mL)
m: Mass of plant sample (g) = 0.5 (as per protocol)
Precautions
1. This kit can also be used with a microplate reader, allowing for a corresponding increase in the number of samples processed. However, a spectrophotometer is preferred if available.
2. The test sample should not contain CAT inhibitors, and repeated freeze-thaw cycles should be avoided.
3. If the CAT Assay Buffer becomes turbid or develops flocculent matter, discard it.
4. The MO Chromogenic Solution is stable for 2 weeks at 4°C after preparation. Discard if a milky-white precipitate forms; prepare fresh is best.
5. For plant samples, grind rapidly to prevent loss of CAT activity, and keep samples on ice during processing. This method is not recommended for plant samples; the Catalase Activity Assay Kit (Ultraviolet Absorption Method) is preferred.
6. CAT in intact red blood cells and undiluted hemolysate is relatively stable for 1 week at 4°C. CAT in diluted hemolysate loses activity easily.
7. Avoid hemolysis caused by sample freezing, as it can decrease catalase activity by 10-15%.
8. Serum CAT activity decreases 64.7% at room temperature in 3 days, 10.5% at 4°C, but only 3.5% after 30 days at -20°C. Store test samples at -20°C or -70°C.
9. For your safety and health, wear lab coats and disposable gloves during operation.
10. Use reagents promptly after opening to avoid affecting subsequent experimental results.
I2623560
危险品化学品经营许可证（带存储）