土壤过氧化氢酶检测试剂盒(S-CAT) 紫外分光光度法

土壤过氧化氢酶（S-CAT）活性检测试剂盒说明书 详见附件
紫外分光光度法
货号：BC0100
规格：50T/24S
产品组成：使用前请认真核对试剂体积与瓶内体积是否一致，有疑问请及时联系索莱宝工作人员。

试剂名称	规格	保存条件
试剂一	液体0.5 mL×1瓶	2-8℃保存
试剂二	粉剂×1瓶	2-8℃保存
试剂三	液体6 mL×1瓶	2-8℃保存

溶液的配制：

1. 试剂一：液体置于试剂瓶内EP管中，使用前需将EP管先离心。临用前取0.05 mL试剂一加入9.95mL蒸馏水稀释待用或者按比例配制。用不完的试剂2-8℃保存1周。
2. 试剂二：临用前加入2 mL蒸馏水充分溶解待用，用不完的试剂2-8℃保存4周。

产品说明：
S-CAT是土壤微生物代谢的重要酶类，在H₂O₂清除系统中具有重要作用。
H₂O₂在240nm下有特征吸收峰，通过测定与土壤反应后溶液在此波长下吸光度的变化，即可反应S-CAT活性的高低。
注意：实验之前建议选择2-3个预期差异大的样本做预实验。如果样本吸光值不在测量范围内建议稀释或者增加样本量进行检测。
需自备的仪器和用品：
紫外分光光度计、台式离心机、水浴锅、可调式移液器、1mL石英比色皿、蒸馏水、研钵、30~50目筛和蒸馏水。
 

• 样本处理（可适当调整待测样本量，具体比例可以参考文献）

新鲜土样自然风干或37℃烘箱风干，过30~50目筛。
二、测定步骤

1. 分光光度计预热30min以上，调节波长至240nm，蒸馏水调零。
2. 加样表（1.5ml EP管）：

试剂名称	测定管	无基质管	无土管
风干土样（g）	0.1	0.1
试剂一（μL）	1000		1000
蒸馏水（μL）		1000
25℃振荡培养20min
试剂二（μL）	25	25	25
混匀8000g，25℃离心5min，取全部上清

 

试剂三（μL）

120

120

120

混匀，240nm处记录各管吸光值A。（每个测定管要设一个无基质管，无土管只要做1-2管）
三、S-CAT活力的计算
单位的定义：每天每g风干土样催化1mmol H₂O₂降解定义为一个酶活力单位。
计算公式：S-CAT（U/g 土样）=[(A无土管-A测定管+A无基质管)×V反总÷（ε×d）×10³]÷W÷T
=18.9×（A无土管-A测定管+A无基质管）
V反总：反应体系总体积，1.145×10^-3L；ε：过氧化氢摩尔消光系数，43.6 L/mol/cm；d：比色皿光径，1cm； T：反应时间，20min=1/72d；W：样本质量，0.1g。
注意事项：
如果吸取的上清仍有部分浑浊，可以在加入试剂三后统一再次进行离心。
相关发表文献：
[1] Hou Q, Wang W, Yang Y, et al. Rhizosphere microbial diversity and community dynamics during potato cultivation[J]. European Journal of Soil Biology, 2020, 98: 103176.
参考文献：
[1] 杨兰芳, 曾巧, 李海波, et al. 紫外分光光度法测定土壤过氧化氢酶活性[J]. 土壤通报, 2011, 42(1):207-210.
[2] Johansson L H, Borg L A H. A spectrophotometric method for determination of catalase activity in small tissue samples[J]. Analytical biochemistry, 1988, 174(1): 331-336.
相关系列产品：
BC0280/BC0285  土壤碱性磷酸酶（S-AKP/ALP）活性检测试剂盒
BC0110/BC0115  土壤多酚氧化酶（S-PPO）活性检测试剂盒
BC0120/BC0125  土壤脲酶（S-UE）活性检测试剂盒
BC0140/BC0145  土壤酸性磷酸酶（S-ACP）活性检测试剂盒

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