谷胱甘肽过氧化物酶（GSH-Px, EC.1.11.1.9）是谷胱甘肽氧化还原循环中催化还原型谷胱甘肽（GSH）氧化的主要酶之一。GSH-Px 不仅能够特异地催化还原型谷胱甘肽与活性氧（ROS）反应，生成氧化型谷胱甘肽（GSSG），从而保护生物膜免受 ROS 的损害，维持细胞的正常功能；而且具有保护肝脏、提高机体免疫力、拮抗有害金属离子对机体的伤害和增加机体抗辐射等能力。GSH-Px 的活性中心是硒半胱氨酸，硒是GSH-Px 的必需部分，测定 GSH-Px 的活力可以作为衡量机体硒水平的一项生化指标。

本试剂盒检测原理是 GSH-Px 催化 H₂O₂ 氧化 GSH 产生 GSSG；谷胱甘肽还原酶（GR）催化 NADPH还原 GSSG 再生成 GSH，同时 NADPH 被氧化成 NADP⁺。测定 NADPH 在 340 nm 处吸光度值的减少速率即可计算样本中 GSH-Px 的活性。

组分货号	组分名称	物理外观	48T	96T	储存
G1527168A	分析缓冲液	液体	70mL	2×70mL	2-8℃.
G1527168B	谷胱甘肽还原酶	液体	5μL	5μL	2-8℃.避光保存
G1527168C	过氧化氢	液体	200μL	200μL	2-8℃.避光保存
G1527168D	底物	固体	1 vial	1 vial	-20℃.避光保存

注：整套试剂盒未拆封于−20 ℃避光保存，自生产之日起有效期 12 个月。拆封后各组分稳定性不同：为避免反复冻融，组分 G1527168A、G1527168B、G1527168C 可转至 2‑8 ℃存放，有效期仍为 12 个月；组分 G1527168D 需继续冷冻保存。

*请在实验前检查各组分的量。

*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

自备资源类型	资源名称	备注
仪器	酶标仪	能测 340 nm 处的吸光度
耗材	96 孔酶标板	普通透明板
试剂	去离子水/PBS	试剂配置用/样本清洗用
其他	匀浆器（组织样本）、低温离心机、恒温箱、制冰机、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. 建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。

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。

试剂准备

试剂名称	试剂准备	注意事项
分析缓冲液	即用型；使用前平衡到室温	4℃保存
底物工作液	临用前配制；底物加入20 mL分析缓冲液，充分溶解	实验过程中冰上避光放置；用不完的试剂-20℃分装避光保存1个月，禁止反复冻融。
谷胱甘肽还原酶	即用型；使用前平衡到室温	实验过程中避光放置；4℃避光保存
H₂O₂工作液	临用前配制；吸取21.5 μL H₂O₂加入5 mL去离子水，充分混匀	4℃避光保存；配制好的试剂当天使用

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 1 个月。样品处理等过程均需要在冰上进行，且须在当日测定酶活力；细胞中 GSH-Px 活性测定时，细胞数目须在 300 万-500 万之间，细胞中 GSH-Px的提取时可加分析缓冲液后研磨或超声波处理，不能用细胞裂解液处理细胞。

1.动植物组织：用冷的 PBS 清洗组织，尽可能去除血液。吸干组织上的水分，称重。称取 0.1 g 组织样本，加入 1 mL 预冷的分析缓冲液，快速冰上匀浆。匀浆后的样本，10000 g，4℃离心 10 min，取上清液，置冰上待测。

2.细胞（菌）样本：收集 5×106 细胞（菌）到离心管内，用预冷的 PBS 清洗细胞（菌），离心后弃上清，加入 1 mL分析缓冲液，冰浴超声波破碎细胞（菌）5 min（功率 20%或 200 W，超声 3 s，间隔 7 s，重复 30 次），然后 10000 g，4℃离心 10 min，取上清液置冰上待测。

3.血清（浆）：直接测定（如需要可用生理盐水稀释不同倍数）。

实验步骤

1. 酶标仪准备：预热 30 min 以上，调节波长到 340 nm。

2. 工作液的配制：临用前配制；根据样本数量，按照 2 mL底物工作液加入 1 μL谷胱甘肽还原酶的比例配制，再加 6 mL的分析缓冲液混匀（当天用完）；测定前将工作液在 37℃（哺乳动物）或 25℃（其他物种）孵育 30 min 以上。

3. 检测体系配制：在 96 孔 UV 板中按照如下方式操作

试剂	空白孔（μL）	测定孔（μL）
去离子水	20	0
样本	0	20
工作液	160	160
H₂O₂工作液	20	20

吸光度检测：混匀后，分别测定第10 s和第10 min10 s 在 340 nm 的吸光度值，分别记为、A1_测定、A 1_空白和 A2_空白和 A2_测定。

结果计算

1. 数据处理

计算

ΔA_测定 = A1_测定 − A2_测定

ΔA_空白 = A1_空白 − A2_空白。

2. 样本 GSH-Px 含量计算

按样本质量计算

酶活单位定义：在 25℃或 37℃温度下，每 g 样品每分钟催化 1 nmol NADPH 氧化定义为 1 个酶活单位。

GSH-Px (U/g) = [ (ΔA_测定 − ΔA_空白) ÷ (ε × d) × V_反总 × 10⁹ ] ÷ ( V_样 ÷ V_样总 × W ) ÷ T = 321.6 × (ΔA_测定 − ΔA_空白) ÷ W

按细胞或细菌数量计算

酶活单位定义：在 25℃或 37℃温度下，每 10⁴ 个细胞或细菌每分钟催化 1 nmol NADPH 氧化定义为 1 个酶活单位。

GSH-Px (U/10⁴) = [ (ΔA_测定 − ΔA_空白) ÷ (ε × d) × V_反总 × 10⁹ ] ÷ ( 500 × V_样 ÷ V_样总 ) ÷ T = 321.6 × (ΔA_测定 − ΔA_空白) ÷ 500

按液体体积计算

酶活单位定义：在 25℃或 37℃温度下，每 mL 液体每分钟催化 1 nmol NADPH 氧化定义为 1 个酶活单位。

GSH-Px (U/mL) = [ (ΔA_测定 − ΔA_空白) ÷ (ε × d) × V_反总 × 10⁹ ] ÷ V_样 ÷ T = 321.6 × (ΔA_测定 − ΔA_空白)

ε：NADPH 摩尔消光系数，6.22×10³ L/mol/cm；

d：96 孔 UV 板光径，0.5 cm；

V_反总：反应体系总体积，200 μL = 2×10⁻⁴ L；

10⁹：1 mol = 1×10⁹ nmol；

Cpr：上清液蛋白浓度（mg/mL）；

W：样品质量，g；

V_样：加入反应体系中上清液体积，20 μL = 2×10⁻² mL；

V_样总：提取液体积，1 mL；

T：反应时间，10 min；

500：细胞或细菌数量，500 万

结果展示

取 0.1 g 大鼠脑，按照测定步骤操作，用 96 孔板进行检测。

测得

ΔA_测定 = A1_测定 − A2_测定 = 0.17 − 0.148 = 0.022， ΔA_空白 = A1_空白 − A2_空白 = 0.106 − 0.1 = 0.006。

按样本质量计算得：GSH-Px (U/g) = 321.6 × (ΔA_测定 − ΔA_空白) ÷ W = 51.46 U/g。

FAQ

检测得到的样本 ΔA _测定过高或者过低怎么办？如果ΔA _测定小于 0.005 可适当加大样本量，再重新进行测定。如果ΔA _测定大于 1.0，样本可用 Assay Buffer 进一步稀释或减少提取用样本量，再重新进行测定。

Glutathione peroxidase (GSH-Px, EC 1.11.1.9) is one of the major enzymes in the glutathione redox cycle that catalyzes the oxidation of reduced glutathione (GSH). GSH-Px not only specifically catalyzes the reaction of reduced glutathione with reactive oxygen species (ROS) to generate oxidized glutathione (GSSG), thereby protecting biological membranes from ROS damage and maintaining normal cellular function, but also possesses the ability to protect the liver, enhance immunity, antagonize the damage caused by harmful metal ions, and increase the body's resistance to radiation. The active site of GSH-Px is selenocysteine, and selenium is an essential component of GSH-Px. Therefore, measuring GSH-Px activity can serve as a biochemical indicator for assessing selenium levels in the body.

The detection principle of this kit is as follows: GSH-Px catalyzes the oxidation of GSH by H₂O₂ to produce GSSG; glutathione reductase (GR) catalyzes the reduction of GSSG back to GSH using NADPH, while NADPH is oxidized to NADP⁺. The GSH-Px activity in the sample can be calculated by measuring the decrease rate of NADPH absorbance at 340 nm.

Component Table

Component No.	Component Name	Appearance	48T	96T	Storage
G1527168A	Assay Buffer	Liquid	70mL	2×70mL	2-8℃.
G1527168B	GR	Liquid	5μL	5μL	2-8℃.Store at dark.
G1527168C	H₂O₂	Liquid	200μL	200μL	2-8℃.Store at dark.
G1527168D	Substrate	Solid	1 vial	1 vial	-20℃.Store at dark.

Note: Store the unopened kit at −20 °C, protected from light. The shelf‑life is 12 months from the date of manufacture. After opening, each component exhibits different stability. To avoid repeated freeze‑thaw cycles, components G1527168A, G1527168B and G1527168C may be stored at 2‑8 °C while retaining the original 12‑month shelf‑life. Component G1527168D shall remain frozen for storage.

*Please check the quantity of each component before the experiment.

*An additional 10% of each component is provided beyond the specifications for standard curve preparation or preliminary experiments.

Reagents, consumables and Equipments not provided

Type	Resource Name	Remarks
Instruments	Microplate reader	Capable of measuring absorbance at 340 nm
Consumables	96‑well microplate	Standard transparent plate
Reagents	Deionized water / PBS	For reagent preparation / sample washing
Others	Homogenizer (for tissue samples), refrigerated centrifuge, incubator, ice maker, adjustable pipettes and tips	Using a multichannel pipette is helpful for improving efficiency when handling a large number of samples

Precautions

1.Before formal testing, it is recommended to perform preliminary experiments using 2–3 samples with expected large differences

2.For tissue and cell samples, result normalization can be achieved by measuring protein concentration.

3.It is recommended that users establish their own standard curve for better accuracy. If not, the typical standard curve formula shown in the Results section can be used for calculation.

4.Biochemical reagents are generally irritating and biologically toxic. For your safety, wear lab coats, masks, gloves, head covers, and other protective equipment. Perform all experiments in a fume hood or biosafety cabinet.

Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Assay Buffer	Ready-to-use; equilibrate to room temperature before use	Store at 4°C
Working Substrate	Prepare immediately before use; add 20 mL of Assay Buffer to the Substrate vial and dissolve thoroughly	Keep on ice and protected from light during the experiment; store unused reagent at -20°C in aliquots, protected from light, for up to 1 month. Avoid repeated freeze-thaw cycles.
GR	Ready-to-use; equilibrate to room temperature before use	Keep protected from light during the experiment; store at 4°C in the dark
Working H₂O₂	Prepare immediately before use; pipette 21.5 μL of H₂O₂ into 5 mL of deionized water and mix thoroughly	Store at 4°C in the dark; use the prepared solution on the same day

Sample Preparation

Note: Fresh samples are recommended. If the experiment is not performed immediately, samples can be stored at -80°C for 1 month. All sample processing steps must be performed on ice, and enzyme activity must be determined on the same day. For GSH-Px activity measurement in cells, the cell number should be between 3 million and 5 million. Cells can be homogenized by grinding or ultrasonic treatment after adding Assay Buffer; do not use cell lysis buffer to treat cells.

1.Animal/Plant Tissues: Wash the tissue with cold PBS to remove blood as much as possible. Blot dry the tissue. Weigh 0.1 g of tissue sample, add 1 mL of pre-cooled Assay Buffer, and homogenize quickly on ice. Centrifuge the homogenate at 10,000 g for 10 min at 4°C. Collect the supernatant and keep it on ice for testing.

2.Cell (or Bacteria) Samples: Collect 5×10⁶ cells (or bacteria) into a centrifuge tube. Wash the cells (or bacteria) with pre-cooled PBS. After centrifugation, discard the supernatant. Add 1 mL of Assay Buffer and disrupt the cells (or bacteria) by ultrasonic treatment on an ice bath for 5 min (power 20% or 200 W, ultrasonic 3 s, interval 7 s, repeat 30 times). Then centrifuge at 10,000 g for 10 min at 4°C. Collect the supernatant and keep it on ice for testing.

3.Serum/Plasma: Measure directly (can be diluted with physiological saline if needed).

Assay Procedure

1.Microplate Reader Preparation: Preheat for more than 30 min and set the wavelength to 340 nm.
2.Working Reagent Preparation: Prepare immediately before use. According to the number of samples, prepare the Working Reagent by adding 1 μL of GR to 2 mL of Working Substrate, then add 6 mL of Assay Buffer and mix well (use on the same day). Before the measurement, incubate the Working Reagent at 37°C (for mammalian samples) or 25°C (for other species) for more than 30 min.
3.Reaction System Setup: In a 96-well UV plate, operate as follows:

Reagent	Blank Well (μL)	Sample Well (μL)
Deionized Water	20	0
Sample	0	20
Working Reagent	160	160
Working H₂O₂	20	20

4.Absorbance Measurement: After mixing, measure the absorbance at 340 nm at 10 seconds and at 10 minutes 10 seconds. Record these values as A1_Sample, A2_Sample, A1_Blank, and A2_Blank, respectively.

Calculation of Results

1.Data Processing Calculate: ΔA_Sample = A1_Sample − A2_Sample ΔA_Blank = A1_Blank − A2_Blank.

2.Calculation of Sample

GSH-Px Content

Calculated by Sample Weight

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute per gram of sample at 25°C or 37°C.

GSH-Px (U/g) = [ (ΔA_Sample − ΔA_Blank) ÷ (ε × d) × V_Total × 10⁹ ] ÷ ( V_Sample ÷ V_{Total Extract} × W ) ÷ T = 321.6 × (ΔA_Sample − ΔA_Blank) ÷ W

Calculated by Cell or Bacteria Number

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute per 10⁴ cells or bacteria at 25°C or 37°C.

GSH-Px (U/10⁴) = [ (ΔA_Sample − ΔA_Blank) ÷ (ε × d) × V_Total × 10⁹ ] ÷ ( 500 × V_Sample ÷ V_{Total Extract} ) ÷ T = 321.6 × (ΔA_Sample − ΔA_Blank) ÷ 500

Calculated by Liquid Volume

Unit definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute per mL of liquid at 25°C or 37°C. GSH-Px (U/mL) = [ (ΔA_Sample − ΔA_Blank) ÷ (ε × d) × V_Total × 10⁹ ] ÷ V_Sample ÷ T = 321.6 × (ΔA_Sample − ΔA_Blank) Where:

ε: Molar extinction coefficient of NADPH, 6.22×10³ L/mol/cm;

d: Light path of the 96-well UV plate, 0.5 cm;

V_Total: Total reaction volume, 200 μL = 2×10⁻⁴ L;

10⁹: 1 mol = 1×10⁹ nmol;

W: Sample weight, g;

V_Sample: Volume of supernatant added to the reaction system, 20 μL = 2×10⁻² mL;

V_{Total Extract}: Extraction volume, 1 mL;

T: Reaction time, 10 min;

500: Cell or bacteria number, 500×10⁴.

FAQ

What if the measured ΔA_Sample is too high or too low? If ΔA_Sample is less than 0.005, the sample volume can be appropriately increased and the assay repeated. If ΔA_Sample is greater than 1.0, the sample can be further diluted with Assay Buffer, or the amount of sample used for extraction can be reduced, and the assay repeated.

规格

规格或纯度

BioReagent

英文名称

Glutathione Peroxidase Activity Assay Kit (NADPH Rate, UV Micro Method)

应用

酶活性检测

检测方法

分光光度法

检测仪器

酶标仪

检测波长

340nm

敏感性

Light Sensitive

储存与运输

储存条件

2-8°C储存,避光,-20°C储存

运输条件

超低温运输

稳定性与储存

各组分在相应的储存条件下保质期12个月。

组分与储存

组分货号	组分名称	物理外观	48T	96T	储存
G1527168A	分析缓冲液	液体	70mL	2×70mL	2-8℃.
G1527168B	谷胱甘肽还原酶	液体	5μL	5μL	2-8℃.避光保存
G1527168C	过氧化氢	液体	200μL	200μL	2-8℃.避光保存
G1527168D	底物	固体	1 vial	1 vial	-20℃.避光保存

注：整套试剂盒未拆封于−20 ℃避光保存，自生产之日起有效期 12 个月。拆封后各组分稳定性不同：为避免反复冻融，组分 G1527168A、G1527168B、G1527168C 可转至 2‑8 ℃存放，有效期仍为 12 个月；组分 G1527168D 需继续冷冻保存。注：整套试剂盒未拆封于−20 ℃避光保存，自生产之日起有效期 12 个月。拆封后各组分稳定性不同：为避免反复冻融，组分 G1527168A、G1527168B、G1527168C 可转至 2‑8 ℃存放，有效期仍为 12 个月；组分 G1527168D 需继续冷冻保存。

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技术文档和文章

g1527168 谷胱甘肽过氧化物酶活性检测试剂盒说明书.pdf

g1527168 glutathione peroxidase activity assay kit nadph rate uv micro method manual.pdf

谷胱甘肽还原酶：维持GSH/GSSG稳态的黄素依赖还原体系及其检测与应用

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探索主题:

NADPH GSH GR 氧化应激 FAD GSSG 谷胱甘肽还原酶 340 nm 偶联测定抗氧化体系

Glutathione Reductase: A Flavin-Dependent Reductive System Maintaining GSH/GSSG Homeostasis, with Assays and Applications

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GSH GR GSSG glutathione reductase

细胞过氧化物清除酶系统：谷胱甘肽过氧化物酶、过氧化氢酶与过氧化还原蛋白的功能分工

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谷胱甘肽过氧化氢酶谷胱甘肽过氧化物酶 GPX4 铁死亡脂质过氧化 Peroxiredoxin

Cellular Peroxide-Scavenging Enzyme Systems: Functional Division of Glutathione Peroxidase, Catalase, and Peroxiredoxin

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探索主题:

glutathione Catalase Glutathione Peroxidase GPX4 ferroptosis lipid peroxidation Peroxiredoxin

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谷胱甘肽过氧化物酶活性检测试剂盒 (NADPH速率, 紫外微量法)

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