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硫氧还蛋白过氧化物酶（Thioredoxin Peroxidase，TPX）（EC 1.11.1.24）属于过氧化物酶家族，在体内主要通过还原过氧化氢和一些氢过氧化物来实现抗氧化作用，功能与谷胱甘肽过氧化物酶（GPX）类似，也是谷胱甘肽氧化还原循环关键酶之一。TPX 普遍存在于各种生物体内，如酵母、植物、动物、原生动物、寄生虫、细菌和古细菌，在进化上高度保守。TPX 与细胞增殖、分化、细胞凋亡及肿瘤发生调控密切相关。TPX 的主要功能包括细胞脱毒、抗氧化和调节由过氧化氢介导的信号转导和免疫反应。
本试剂盒检测原理是 TPX 能够催化 H₂O₂氧化底物二硫苏糖醇（DTT）。在反应过程中，底物 H₂O₂在 240 nm波长处具有特征吸收峰；随着酶促反应的进行，H₂O₂被不断消耗，导致该波长下的吸光度值随之下降。通过测定 240 nm 吸光度的下降速率，测定样品 TPX 活性。
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注意：
1.请在实验前检查各组分的量。
2.各组分在规格基础上额外提供 10%的量用于进行标准曲线制作或预实验。
自备仪器与试剂
1. 仪器：酶标仪（能测 240 nm 处的吸光度）
2. 耗材：96孔酶标板（UV 板）
3. 试剂：PBS（样本清洗用）
4. 其他：匀浆器（组织样本）、制冰机、水浴锅、低温离心机、可调节式移液枪及枪头（检测量较大时合理使用多通道移液器有助于提高实验效率）
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.对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。
3.本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4.生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
5.本产品仅供科学研究使用，不适用于临床诊断。
实验流程
1 试剂准备
检测缓冲液，底物以及过氧化氢在使用前平衡至室温。
注意： 过氧化氢有一定的刺激性，建议在使用过程中做好个人防护。
2 样本制备
动物组织：称取约 0.1 g 样本，加入 1 m 检测缓冲液，冰浴匀浆，8000 g，4℃离心 10 min，取上清置冰上待测。
细胞或细菌：收集 5×10⁶个细胞或细菌，用预冷的PBS清洗细胞或细菌，800 g 离心 2 min，弃上清。加入1 mL 检测缓冲液进行冰浴超声破碎。超声波破碎 5 min（功率 20%或 200 W，超声 3 s，间隔 7 s，重复30 次），8000 g，4℃离心 10 min，取上清置于冰上待测。
血清（浆）：直接测定。
3 实验步骤
1. 酶标仪准备：预热 30 min 以上，调节波长到 240 nm。
2. 检测前，底物置于 25℃（普通物种）或者 37℃（哺乳动物）中预热 30 min 以上。
3. 检测体系配制：在 96 孔 UV 板中加样，每孔先加入 4 μL 上清液，再加入 180 μL 底物，最后加入 16 μL过氧化氢，轻柔晃动孔板，使各组分混合均匀。
4. 吸光度检测：测定 240 nm 处的吸光值，分别记录 10 s 和 2 min 10 s 吸光值，记为 A1 和 A2 。
结果计算
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
1. 数据处理
计算 ΔA测定=A1−A2
2. 样本 TPX 活性计算
● 按蛋白浓度计算
活性单位 (U) 定义：25℃或者 37℃中，每毫克蛋白每分钟催化 1 nmol过氧化氢降解。
TPX 活性 (U/mg prot)=ΔA测定÷(ε×d)×V反总÷(Cpr×V样)÷T=1147×ΔA测定÷Cpr
● 按样本鲜重计算
活性单位 (U) 定义：25℃或者 37℃中，每克样品每分钟催化 1 nmol过氧化氢降解。
TPX 活性 (U/g)=ΔA测定÷(ε×d)×V反总÷(V样÷V样总×W)÷T=1147×ΔA测定÷W
● 按细胞或细菌数量计算
活性单位 (U) 定义：25℃或者 37℃中，每 10⁴个细胞或细菌每分钟催化 1 nmol过氧化氢降解。
TPX 活性 (U/10⁴)=ΔA测定÷(ε×d)×V反总÷(N×V样÷V样总)÷T=1147×ΔA测定÷N
● 按液体体积计算
活性单位 (U) 定义：25℃或者 37℃中，每毫升液体每分钟催化 1 nmol过氧化氢降解。
TPX 活性 (U/mL)=ΔA测定÷(ε×d)×V反总÷V样÷T=1147×ΔA测定
结果展示 ε：过氧化氢摩尔消光系数，43600 L/mol/cm=0.0436 mL/nmol/cm；d：96 孔 UV 板光径，0.5 cm；V反总：反应体系总体积，200 μL=0.2 mL；Cpr：蛋白浓度，mg/mL；W：样品质量，g；V样：加入反应体系上清体积，4 μL=4×10⁻³ mL；V样总：提取缓冲液体积，1 mL；T：反应时间，2 min；N：细胞 / 细菌数量，以 10⁴计（如细胞总数 5×10⁶，则 N=500）。
示例-1: 取 0.1 g 小鼠肝脏，96 孔 UV 板检测，ΔA测定=A1−A2=1.094−0.995=0.101，代入公式 y=196.995，按组织质量计算：TPX(U/g)=1147×ΔA测定÷0.1=1158.47 U/g。
示例-2: 取 0.1 g 小鼠肾脏，96 孔 UV 板检测，ΔA测定=A1−A2=0.673−0.606=0.067，代入公式得：TPX (U/g)=1147×ΔA测定÷0.1=768.49 U/g。
FAQ
检测得到的样本 ΔA测定过高或者过低怎么办？
若样本 ΔA测定＞0.4，采用提取缓冲液稀释样本或减少提取样本用量后重测；若 ΔA测定＜0.002，可适当增加样本用量。
Thioredoxin Peroxidase (TPX, EC 1.11.1.24) belongs to the peroxidase family. It exerts antioxidant effects mainly by reducing hydrogen peroxide and certain hydroperoxides in vivo. Its function is similar to glutathione peroxidase (GPX), and it is also one of the key enzymes in the glutathione redox cycle. TPX is ubiquitously distributed in all types of organisms, including yeasts, plants, animals, protozoa, parasites, bacteria and archaea, and it is highly conserved during evolution. TPX is closely associated with the regulation of cell proliferation, differentiation, apoptosis and tumorigenesis. Its primary biological functions include cellular detoxification, antioxidant defense, and modulation of hydrogen peroxide-mediated signal transduction and immune responses.
The detection principle of this kit is as follows: TPX catalyzes the oxidation of the substrate dithiothreitol (DTT) by H₂O₂. The substrate H₂O₂ has a characteristic absorption peak at 240 nm. As the enzymatic reaction proceeds, H₂O₂ is continuously consumed, leading to a decline in absorbance at this wavelength. The TPX activity of samples is quantified by measuring the decreasing rate of absorbance at 240 nm.
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Notes：
1.Check the volume of each component before the experiment.
2.An extra 10% volume of each component is supplied on top of the labeled specification for standard curve preparation or pre-experiments.
Self-supplied Instruments and Reagents
1.Instruments: Microplate reader capable of measuring absorbance at 240 nm
2.Consumables: 96-well UV microplate
3.Reagent: PBS (for sample washing)
4.Others: Tissue homogenizer (for tissue samples), ice maker, water bath, refrigerated centrifuge, adjustable pipettes and pipette tips. Multi-channel pipettes are recommended to improve experimental efficiency for large batches of samples.
Precautions
1.Perform pre-experiments with 2–3 samples showing expected large differences prior to formal detection.
2.For tissue and cell samples, protein concentration measurement can be used to normalize results between different samples.
3.This kit is compatible with spectrophotometer detection; proportionally adjust the preparation volume of detection reagents according to spectrophotometer requirements.
4.Biochemical detection reagents are generally irritant and biologically toxic. For your safety and health, complete personal biosafety protection throughout the experiment, including lab coat, mask, gloves and hairnet. Conduct all operations in a fume hood or biosafety cabinet.
5.This product is for research use only and not intended for clinical diagnosis.
Experimental Procedures
1. Reagent Preparation
Equilibrate Assay Buffer, Substrate and H₂O₂ to room temperature before use.
Notes: Substrate is irritant; proper personal protection is required during handling.
2. Sample Preparation
Animal tissues: Weigh approximately 0.1 g tissue sample, add 1 mL Assay Buffer, homogenize on ice. Centrifuge at 8000 g, 4 °C for 10 min, collect supernatant and store on ice for testing.
Cells or bacteria: Collect 5×10⁶ cells or bacteria, wash with pre-cooled PBS, centrifuge at 800 g for 2 min and discard supernatant. Add 1 mL Assay Buffer and lyse on ice by sonication for 5 min (power: 20% / 200 W, sonicate for 3 s with 7 s interval, repeat 30 times). Centrifuge at 8000 g, 4 °C for 10 min, collect supernatant and store on ice for testing.
Serum (plasma): Detect directly without pretreatment.
3. Assay Steps
(1) Prepare the microplate reader: Preheat for more than 30 min and set the wavelength to 240 nm.
(2) Preheat Substrate at 25 °C (for general species) or 37 °C (for mammals) for at least 30 min before detection.
(3) Prepare reaction system in a 96-well UV microplate: Add 4 μL supernatant to each well first, followed by 180 μL Substrate, then 16 μL H₂O₂. Gently shake the plate to fully mix all components.
(4) Absorbance measurement: Record absorbance values at 240 nm at 10 s and 2 min 10 s, designated as A1 and A2 respectively.
Result Calculation
Two equivalent formulas are provided below: a full derivation formula and a simplified concise formula.
1. Data processing
Calculate ΔAsample = A1 − A2
2. Calculation of sample TPX activity
● Calculation based on protein concentration
● Calculation based on fresh sample weight Definition of activity unit (U): At 25 °C or 37 °C, one unit (U) refers to the amount of enzyme that catalyzes the degradation of 1 nmol H₂O₂ per minute per milligram of protein.
TPX activity (U/mg prot) = ΔAsample ÷ (ε × d) × Vtotal ÷ (Cpr × Vsample) ÷ T = 1147 × ΔAsample ÷ Cpr
Definition of activity unit (U): At 25 °C or 37 °C, one unit (U) refers to the amount of enzyme that catalyzes the degradation of 1 nmol H₂O₂ per minute per gram of sample.
TPX activity (U/g) = ΔAsample ÷ (ε × d) × Vtotal ÷ (Vsample ÷ Vtotal-extract × W) ÷ T = 1147 × ΔAsample ÷ W
● Calculation based on cell/bacterial count
Definition of activity unit (U): At 25 °C or 37 °C, one unit (U) refers to the amount of enzyme that catalyzes the degradation of 1 nmol H₂O₂ per minute per 10⁴ cells or bacteria.
TPX activity (U/10⁴ cells) = ΔAsample ÷ (ε × d) × Vtotal ÷ (N × Vsample ÷ Vtotal-extract) ÷ T = 1147 × ΔAsample ÷ N
● Calculation based on liquid volume
Definition of activity unit (U): At 25 °C or 37 °C, one unit (U) refers to the amount of enzyme that catalyzes the degradation of 1 nmol H₂O₂ per minute per milliliter liquid sample.
TPX activity (U/mL) = ΔAsample ÷ (ε × d) × Vtotal ÷ Vsample ÷ T = 1147 × ΔAsample
Parameter explanation:
ε: Molar extinction coefficient of H₂O₂, 43600 L/mol/cm = 0.0436 mL/nmol/cm
d: Light path of 96-well UV plate, 0.5 cm
V<sub>total</sub>: Total volume of reaction system, 200 μL = 0.2 mL
Cpr: Protein concentration of supernatant, mg/mL
W: Mass of sample, g
V<sub>sample</sub> : Volume of supernatant added to reaction system, 4 μL = 4×10⁻³ mL
V<sub>total extract</sub> : Total volume of extraction buffer, 1 mL
T: Reaction time, 2 min
N: Cell or bacterial count in units of 10⁴ (e.g., for 5×10⁶ cells, N = 500)
Result Demonstration
Example 1: Take 0.1 g mouse liver and operate according to the assay protocol using a 96-well UV microplate. Measured ΔAsample = A1 − A2 = 1.096 − 0.995 = 0.101. Calculated activity based on sample weight: TPX activity (U/g) = 1147 × ΔAsample ÷ W = 1158.47 U/g.
Example 2: Take 0.1 g mouse kidney and operate according to the assay protocol using a 96-well UV microplate. Measured ΔAsample = A1 − A2 = 0.673 − 0.606 = 0.067. Calculated activity based on sample weight: TPX activity (U/g) = 1147 × ΔAsample ÷ W = 768.49 U/g.
FAQ
What should I do if the measured ΔA<sub>sample</sub> of the sample is too high or too low?
If ΔAsample < 0.002, appropriately increase the sample loading amount. If ΔAsample > 0.4, dilute the sample further with Extraction Buffer or reduce the initial sample mass during extraction, then re-test.
| Item No. | Appearance | Components | 48T | 96T | Storage |
| T1515798A | 液体 | 检测缓冲液 | 60 mL | 60 mL×2 | 2-8℃ |
| T1515798B | 液体 | 底物 | 11 mL | 22 mL | -20℃. Store in the dark. |
| T1515798C | 液体 | 过氧化氢 | 1 mL | 2 mL | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939442 |
分析证书 | T1515798 |
| ZJ26F0939441 |
分析证书 | T1515798 |
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