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GST是一种具有多种生理功能的蛋白质家族，主要存在于细胞质内。GST是体内解毒酶系统的重要组成部分，主要催化各种化学物质及其代谢产物与GSH的巯基共价结合，使亲电化合物变为亲水物质，易于从胆汁或尿液中排泄，达到将体内各种潜在或具备毒性的物质降解并排出体外的目的。因此，GST在保护细胞免受亲电子化合物的损伤中发挥着重要的生物学功能。此外，因为GST具有GSH-Px活性，亦称为non-Se GSH-Px，具有修复氧化破坏的大分子如DNA、蛋白质等的功能。注意，GST催化的反应减少GSH含量，但是不增加GSSG含量。
测定原理：GST催化GSH与CDNB结合，其结合产物的光吸收峰波长为340nm；通过测定340nm波长处吸光度上升速率，即可计算出GST活性。
注：正式测定前务必取2-3个预期差异较大的样本做预测定
组分与储存：
注：试剂三临用前取一管加入3mL蒸馏水，溶解备用，用不完的试剂可-20℃根据实验用量分装保存，禁止反复冻融两周内使用完，若溶液变成淡黄色或黄色，不可再使用。 |
自备仪器和试剂：
⁕ 紫外分光光度计 (能测340nm处的吸光度)
⁕ 水浴锅、冰盒、低温离心机、1mL石英比色皿、可调式移液器
⁕ 蒸馏水
样本处理：
1. 组织：按照组织质量 (g)：试剂一体积 (mL) 为1：5~10的比例 (建议称取约0.1g组织，加入1mL试剂一)进行冰浴匀浆。8000g，4℃离心10min，取上清置冰上待测。
2. 细菌、真菌：按照细胞数量 (10⁴个)：试剂一体积 (mL) 为500~1000：1的比例 (建议500万细胞加入1mL试剂一)，冰浴超声波破碎细胞 (功率300w，超声3秒，间隔7秒，总时间3min)；然后8000g，4℃，离心10min，取上清置于冰上待测。
3. 血清等液体：直接测定。
测定步骤：
1. 紫外分光光度计预热30 min以上，调节波长至340 nm，蒸馏水调零。
2. 试剂二在37℃水浴中保温30 min。
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计算：
1. 按蛋白浓度计算
活性单位定义：在37℃中条件下，每毫克蛋白每分钟催化1µmol/L CDNB与GSH结合为1个酶活单位。
GST (U/mg prot)=ΔA÷ε÷d×10⁶×V反总÷(Cpr×V样)÷T=0.23×ΔA÷Cpr
2. 按样本质量计算
活性单位定义：
在37℃中条件下，每克样本每分钟催化1µmol/L CDNB与GSH结合为1个酶活单位。
GST (U/g鲜重)=ΔA÷ε÷d×10⁶×V反总÷(W×V样÷V样总)÷T=0.23×ΔA÷W
3. 按细胞数量计算 活性单位定义：
在37℃中条件下，每10⁴个细胞每分钟催化1µmol/L CDNB与GSH结合为1个酶活单位。
GST (U/10⁴ cell)=ΔA÷ε÷d×10⁶×V反总÷(细胞数量×V样÷V样总)÷T=0.23×ΔA÷细胞数量
4. 按液体体积计算 活性单位定义：
在37℃中条件下，每毫升样本每分钟催化1µmol/L CDNB与GSH结合为1个酶活单位。
GST (U/mL)=ΔA÷ε÷d×10⁶×V反总÷V样÷T=0.23×ΔA
ε：产物摩尔消光系数，9.6×10³ L/mol/cm
d：石英比色皿光径，1cm
10⁶：1mol=1×10⁶μmol
V反总：反应体系总体积，1100μl=0.0011 L
Cpr：上清液蛋白质浓度 (mg/mL)
V样：加入反应体系中上清液体积，100μl=0.1 mL
V样总：加入提取液体积，1mL
W，样本质量，g
T：反应时间 (min)，5min
W：样本质量，g
细胞数量：以10⁴为单位，万
注意事项：
1. 若A1>1.0，则需要将样本用试剂一稀释再进行测定，计算公式注意乘以稀释倍数。
2．A1≥A2，说明样本活力太低，需增加样本浓度，或者延长反应时间，计算公式也要做出相应改变。
3. 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。
4. 细胞中GST活性测定时，细胞数目须在300万-500万之间，细胞中GST的提取时可加试剂一后研磨或超声波处理，不能用细胞裂解液处理细胞。
5. 测定反应的温度对测定结果有影响，请控制在37℃。
实验实例：
称取牛肝0.1g，加入1mL提取液，按照上述步骤操作，测得结果如下：
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常见问题及解决方案：
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Glutathione S-transferase (GST) is a family of proteins with diverse physiological functions, predominantly present in the cytoplasm. As a critical component of the body’s detoxification enzyme system, GST mainly catalyzes the covalent conjugation of various chemicals and their metabolites with the sulfhydryl group of glutathione (GSH). This reaction converts electrophilic compounds into hydrophilic substances that are readily excreted via bile or urine, thereby achieving the degradation and elimination of various potentially or intrinsically toxic substances from the body. Accordingly, GST exerts important biological functions in protecting cells from damage caused by electrophilic compounds. In addition, since GST possesses glutathione peroxidase (GSH-Px) activity, it is also referred to as non-selenium GSH-Px (non-Se GSH-Px), and has the function of repairing oxidatively damaged macromolecules such as DNA and proteins. Note that the reaction catalyzed by GST reduces GSH content but does not increase the content of oxidized glutathione (GSSG).
Assay Principle: GST catalyzes the conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB), and the resulting conjugate has an absorption peak at a wavelength of 340 nm. The GST activity can be calculated by measuring the rate of absorbance increase at 340 nm.
Note: It is imperative to perform a pilot assay on 2–3 samples expected to show significant differences prior to the formal measurement.
Contents & Storage:
Reagent 3: Prior to use, add 3 mL of distilled water to one vial and dissolve thoroughly for use. Unused portions can be aliquoted according to experimental needs and stored at -20 °C. Repeated freeze-thaw cycles are strictly prohibited, and the reagent must be used up within two weeks. If the solution turns pale yellow or yellow, it must not be used. |
Instruments and Reagents to Be Prepared by User:
⁕ UV spectrophotometer (capable of measuring absorbance at 340 nm)
⁕ Water bath, ice box, refrigerated centrifuge, 1 mL quartz cuvettes, adjustable pipettes
⁕ Distilled water
Sample Preparation:
1. Tissue samples: Homogenize in an ice bath at a ratio of tissue mass (g) to Reagent 1 volume (mL) of 1:5–10 (it is recommended to weigh approximately 0.1 g of tissue and add 1 mL of Reagent 1). Centrifuge at 8000 g and 4 °C for 10 min, then collect the supernatant and keep it on ice for subsequent assay.
2. Bacterial and fungal samples: Disrupt cells by sonication in an ice bath at a ratio of cell count (10⁴ cells) to Reagent 1 volume (mL) of 500–1000:1 (it is recommended to add 1 mL of Reagent 1 to 5×10⁶ cells). Sonication parameters: power 300 W, 3 s sonication with 7 s interval, total duration 3 min. Then centrifuge at 8000 g and 4 °C for 10 min, collect the supernatant and keep it on ice for assay.
3. Liquid samples (e.g. serum): Can be assayed directly.
Assay Procedure:
1. Preheat the UV spectrophotometer for at least 30 min, set the wavelength to 340 nm, and calibrate the baseline to zero with distilled water.
2. Incubate Reagent 2 in a 37 °C water bath for 30 min.
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Calculations:
1. Calculation based on protein concentration
Unit definition: At 37 °C, one unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the conjugation of 1 µmol/L CDNB with GSH per milligram of protein per minute.
GST (U/mg prot) = ΔA ÷ ε ÷ d × 10⁶ × Vtotal reaction ÷ (Cpr × Vsample) ÷ T = 0.23 × ΔA ÷ Cpr
2. Calculation based on sample mass
Unit definition: At 37 °C, one unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the conjugation of 1 µmol/L CDNB with GSH per gram of sample per minute.
GST (U/g fresh weight) = ΔA ÷ ε ÷ d × 10⁶ × Vtotal reaction ÷ (W × Vsample ÷ Vtotal extract) ÷ T = 0.23 × ΔA ÷ W
3. Calculation based on cell count
Unit definition: At 37 °C, one unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the conjugation of 1 µmol/L CDNB with GSH per 10⁴ cells per minute. GST (U/10⁴ cells) = ΔA ÷ ε ÷ d × 10⁶ × Vtotal reaction ÷ (cell count × Vsample ÷ Vtotal extract) ÷ T = 0.23 × ΔA ÷ cell count
4. Calculation based on liquid sample volume
Unit definition: At 37 °C, one unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the conjugation of 1 µmol/L CDNB with GSH per milliliter of sample per minute.
GST (U/mL) = ΔA ÷ ε ÷ d × 10⁶ × Vtotal reaction ÷ Vsample ÷ T = 0.23 × ΔA
ε: Molar extinction coefficient of the reaction product, 9.6 × 10³ L/mol/cm
d: Path length of the quartz cuvette, 1 cm
10⁶: Unit conversion coefficient, 1 mol = 1 × 10⁶ μmol
Vtotal reaction: Total volume of the reaction system, 1100 μL = 0.0011 L
Cpr: Protein concentration of the supernatant, mg/mL
Vsample: Volume of supernatant added into the reaction system, 100 μL = 0.1 mL
Vtotal extract: Volume of extraction solution added, 1 mL
W: Sample mass, g
T: Reaction time, 5 min
Cell count: Calculated in units of 10⁴ cells (ten thousand cells)
Precautions:
1. If A₁ > 1.0, dilute the sample with Reagent 1 before measurement, and multiply the result by the dilution factor in the calculation formula.
2. If A₁ ≥ A₂, it indicates that the enzyme activity of the sample is too low. Increase the sample concentration or extend the reaction time, and adjust the calculation formula accordingly.
3. All procedures including sample preparation must be carried out on ice, and the enzyme activity assay must be completed on the same day.
4. For GST activity assay in cells, the cell count should be between 3×10⁶ and 5×10⁶. For GST extraction from cells, add Reagent 1 followed by grinding or sonication; cell lysis buffer must not be used to treat cells.
5. The reaction temperature affects the assay results. Please maintain the temperature at 37 °C.
Experimental Example:
Weigh 0.1 g of bovine liver, add 1 mL of extraction solution, and perform the assay following the above procedures. The measurement results are as follows:
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Frequently Asked Questions and Solutions:
| Problem | Possible Cause | Solution |
| Poor reproducibility of sample readings | Inappropriate sample type. | Refer to the instruction manual for incompatible sample types. |
| Insufficient homogenization of tissue samples. | Extend sonication time / increase the number of grinding cycles with the homogenizer. | |
| Excessive freeze-thaw cycles of samples. | Aliquot samples to avoid repeated freeze-thaw cycles. | |
| Interfering substances present in samples. | Identify interfering substances and perform deproteinization on samples if necessary. | |
| Sample readings exceed the linear range. | Concentrate or dilute the sample to keep readings within the linear range. | |
| Air bubbles in the wells. | Air bubbles will interfere with readings. Avoid bubble formation during operation and be sure to remove air bubbles before taking readings. | |
| Low/high readings for samples and standards | Samples left for too long or stored improperly. | Use freshly prepared samples and store at the recommended temperature until use. |
| Kit components are not completely thawed. | Mix gently after the components are fully thawed before use. | |
| Reagents left on ice for too long. | Prepare the reaction mixture freshly before each use. | |
| Incorrect incubation time/temperature. | Refer to the incubation time and/or temperature recommended in the instruction manual. | |
| Incorrect reagent pipetting volume. | Check if the pipette is calibrated (always use the smallest-range pipette that can aspirate the full volume); avoid aspirating excessively small volumes of reagents as much as possible. | |
| Pipetting errors during reaction mixture preparation. | Always prepare a master mix. |
| 组分货号 | 组分名称 | 物理外观 | 50T | 储存 |
| G1515910A | 试剂一 | 液体 | 50 mL | 2-8℃. |
| G1515910B | 试剂二 | 液体 | 45 mL | 2-8℃. |
| G1515910C | 试剂三 | 粉末 | 2 vial | 2-8°C. 避光 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0938875 |
分析证书 | G1515910 |
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