土壤过氧化氢酶（S-CAT）主要来源于土壤微生物以及植物根系的分泌物，是土壤生物代谢的重要酶类，主要功能是解除土壤中过氧化氢的毒害作用，在活性氧清除系统中具有重要作用。
检测原理：H₂O₂在240nm处具有特征吸收峰，土壤过氧化氢酶能够分解H₂O₂，使反应溶液在240nm处吸光值随反应时间而下降，根据吸光值变化速率即可表征土壤过氧化氢酶的活性。
S1505477
| Component
| 48T
| 96T
| Storage
|
S1505477A
| Reagent Ⅰ
| 350 μL
| 700 μL
| 2-8℃
|
S1505477B
| Reagent Ⅱ
| 1EA | 1EA
| 2-8℃
|
S1505477C
| Reagent Ⅲ
| 3 mL
| 6 mL
| 2-8℃ |
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
1、酶标仪或紫外分光光度计（能测240nm处的吸光度）
2、96孔UV板或微量石英比色皿、可调节式移液枪及枪头
3、恒温水浴锅、恒温摇床、台式离心机、30-50目筛
4、去离子水
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
Reagent Ⅰ
| 临用前配制，
48T加入34.65mL 去离子水，96T加入69.3mL 去离子水，充分混匀待用；
| 4℃保存1周 |
Reagent Ⅱ
| 临用前配制，
48T加入1.2mL 去离子水，96T加入2.4mL 去离子水，充分溶解待用；
| 4℃保存4周（若有结晶析出，60℃-90℃水浴溶解后使用） |
Reagent Ⅲ
| 即用型；使用前平衡到室温 | 4℃保存 |
2、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存数周。测定时，应控制解冻的温度和时间。室温环境下解冻 时，需在4h内完成样品解冻。
土壤样本：新鲜土样自然风干或37℃烘箱风干，过30-50目筛。
3、实验步骤
3.1 酶标仪或紫外分光光度计预热30min，调节波长到240nm。紫外分光光度计用去离子水调零。
3.2 操作表（下述操作在1.5mL EP管中进行）：
试剂
| 测定管
| 无基质管
| 无土管
|
风干土样 (g)
| 0.03 | 0.03
| 0 |
Reagent Ⅰ (μL)
| 260 | 0
| 260
|
去离子水 (μL)
| 0
| 260
| 0 |
25℃振荡培养20min；
混匀，8,000g，25℃离心5min，取180μL上清至96孔UV板或微量石英比色皿中；
3.3 充分混匀，于240nm处测定各管吸光度，分别记为A测定、A无基质、A无土。
注意：无土管只需测定1次，每个测定管需设置一个无基质管。实验之前建议选择2-3个预期差异大的样本做预实验。如果（A无土-A测定）小于0.01可适当加大样本量。如果（A无土-A测定）大于1.5，减少提取用样本量。
4、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
4.1 用96孔UV板测定的计算公式如下：
单位的定义：每天每g风干土样催化1μmol H₂O₂降解定义为一个酶活力单位。
S-CAT (U/g 土样)=[(A无土-A测定+A无基质)×V反总÷(ε×d)×10⁶]÷W÷T=33×(A无土-A测定+A无基质)
参数说明：
V反总：反应体系总体积，3×10⁻⁴ L；
ε：过氧化氢摩尔消光系数，4.36×10⁴ L/mol/cm；
d：96孔UV板光径，0.5cm；
T：反应时间，20min=1/72 d；
W：样本质量，0.03g。
4.2 用微量石英比色皿测定的计算公式如下：
单位的定义：每天每g风干土样催化1μmol H₂O₂降解定义为一个酶活力单位。
S-CAT (U/g 土样)=[(A无土-A测定+A无基质)×V反总÷(ε×d)×10⁶]÷W÷T=16.5×(A无土-A测定+A无基质)
参数说明：
V反总：反应体系总体积，3×10⁻⁴ L；
ε：过氧化氢摩尔消光系数，4.36×10⁴ L/mol/cm；
d：比色皿光径，1cm；
T：反应时间，20min=1/72 d；
W：样本质量，0.03g。
5、结果展示
图1：本试剂盒测定土壤中过氧化氢酶的活性
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Soil catalase (S-CAT) primarily originates from soil microorganisms and plant root exudates. It is an important enzyme in soil metabolic processes, whose main function is to detoxify hydrogen peroxide in the soil, playing a crucial role in the reactive oxygen species scavenging system.
Assay Principle
H₂O₂ has a characteristic absorption peak at 240 nm. Soil catalase decomposes H₂O₂, causing the absorbance of the reaction solution at 240 nm to decrease over time. The rate of change in absorbance is used to represent the activity of soil catalase.
S1505477
| Component
| 48T
| 96T
| Storage
|
S1505477A
| Reagent Ⅰ
| 350 μL
| 700 μL
| 2-8℃
|
S1505477B
| Reagent Ⅱ
| 1EA | 1EA
| 2-8℃
|
S1505477C
| Reagent Ⅲ
| 3 mL
| 6 mL
| 2-8℃ |
Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.
Required Materials Not Provided
1. Microplate reader or UV spectrophotometer (capable of measuring absorbance at 240 nm)
2. 96-well UV plate or micro quartz cuvettes, adjustable pipettes and tips
3. Constant temperature water bath, incubator shaker, benchtop centrifuge, 30-50 mesh sieve
4. Deionized water
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation Steps
| Notes & Storage
|
Reagent Ⅰ
| Reconstitute before use: Add 34.65 mL (48T) or 69.3 mL (96T) deionized water. Mix thoroughly.
| Stable at 4°C for 1 week.
|
Reagent Ⅱ
| Reconstitute before use: Add 1.2 mL (48T) or 2.4 mL (96T) deionized water. Dissolve completely.
| Stable at 4°C for 4 weeks. If crystals form, dissolve in a 60-90°C water bath before use.
|
Reagent Ⅲ
| Ready-to-use; equilibrate to room temperature before use.
| Store at 4°C. |
2. Sample Preparation
Soil Sample: Air-dry fresh soil samples naturally or in a 37°C oven. Pass through a 30-50 mesh sieve.
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for several weeks. Control thawing temperature and time during assay (if frozen). If thawing at room temperature, complete within 4 hours.
3. Assay Procedure
3.1 Instrument Preparation
Preheat the microplate reader or UV spectrophotometer for 30 minutes. Set the wavelength to 240 nm.
Zero the UV spectrophotometer with deionized water.
3.2 Assay Setup (Perform in 1.5 mL EP tubes)
Reagent
| Test Tube
| No-Substrate Control Tube
| No-Soil Control Tube
|
Air-dried Soil (g)
| 0.03 | 0.03
| 0 |
Reagent Ⅰ (μL)
| 260 | 0
| 260
|
Deionized Water (μL)
| 0
| 260
| 0 |
Incubate at 25°C with shaking for 20 minutes.
Mix well, centrifuge at 8,000 g, 25°C for 5 min. Transfer 180 μL supernatant to a 96-well UV plate or micro quartz cuvette.
3.3 Measurement
Mix thoroughly and measure the absorbance at 240 nm for each tube.
Record the values as ATest, ANo-Substrate, and ANo-Soil.
Note: The No-Soil Control tube only needs to be measured once. Each Test tube requires a corresponding No-Substrate Control tube.
A pilot experiment with 2-3 variable samples is recommended before the formal assay.
If (ANo-Soil - ATest) is less than 0.01, consider increasing the sample amount.
If (ANo-Soil - ATest) is greater than 1.5, reduce the sample amount used for extraction.
4. Calculation of Results
Note: The following derived and simplified formulas are equivalent. The simplified formula (in bold) is recommended as the final calculation method.
Definition of Unit: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the degradation of 1 μmol of H₂O₂ per day per gram of air-dried soil.
4.1 Calculation using a 96-well UV Plate:
S-CAT (U/g soil) = 33 × (ANo-Soil - ATest + ANo-Substrate)
Parameter Definitions:
Vtotal: Total reaction system volume, 3 × 10⁻⁴ L
ε: Molar extinction coefficient of H₂O₂, 4.36 × 10⁴ L/mol/cm
d: Light path of the 96-well UV plate, 0.5 cm
T: Reaction time, 20 min = 1/72 day
W: Sample mass, 0.03 g
4.2 Calculation using a Micro Quartz Cuvette:
S-CAT (U/g soil) = 16.5 × (ANo-Soil - ATest + ANo-Substrate)
Parameter Definitions:
Vtotal: Total reaction system volume, 3 × 10⁻⁴ L
ε: Molar extinction coefficient of H₂O₂, 4.36 × 10⁴ L/mol/cm
d: Light path of the cuvette, 1 cm
T: Reaction time, 20 min = 1/72 day
W: Sample mass, 0.03 g
5. Results Presentation
Figure 1: Typical results showing soil catalase activity measured with this kit
Precautions
1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.
2. This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.
ZJ25F1129758
危险品化学品经营许可证（带存储）