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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
超氧化物岐化酶 (Superoxide Dismutase, SOD) 是含金属辅基的酶，能催化超氧化物阴离子发生岐化作用，生成过氧化氢 (H₂O₂) 和氧气 (O₂)，是生物体内一种重要的抗氧化酶，由于超氧自由基是不稳定的自由基，寿命极短，SOD 活性一般用间接方法测定，并利用各种呈色反应来测定 SOD 活力，其中显色剂有 NBT (四氮唑蓝)、WST-1、WST-8 等。
总超氧化物岐化酶 (SOD) 检测试剂盒 (邻苯三酚比色法) 也叫邻 / 连苯三酚自氧化法或邻 / 连苯三酚自氧化抑制法，其检测原理是邻苯三酚在碱性条件下，能迅速自氧化，释放出超氧阴离子自由基，进而生成黄色的中间产物；在自氧化过程的初始阶段，黄色产物的积累在滞后 30~45s 后与时间呈线性关系，黄色产物在 325nm 处有强吸收，在有 SOD 存在时，由于 SOD 能催化超氧阴离子自由基与氢离子结合成氧气和过氧化氢，从而阻止了中间产物的积累，因此可根据 SOD 抑制邻苯三酚自氧化能力测定 SOD 的酶活力。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
产品组成：
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自备材料：
1、蒸馏水、生理盐水或磷酸缓冲液；
2、离心机、离心管、水浴锅或恒温箱、小试管、分光光度计、石英比色杯。
操作步骤（仅供参考）：
1、 准备样品：
①血浆或含红细胞的样品：如果测定血浆中SDO活性，则从待测样品中分理出血清或血浆，不应有溶血，如果含有红细胞应先4℃ 3000r/min离心5min，转移上清至另一新的离心管中，适量生理盐水稀释后待测，如超过检测范围，用磷酸缓冲液(pH7.8)稀释后再测。如果需要测定红细胞中SOD活性，则应取一定体积的新鲜血液或肝素抗凝血，混匀，3000r/min离心5min，弃上清，沉淀用ACK红细胞裂解液使其充分溶血，3000r/min离心5min，上清液即为红细胞SOD粗提液。
②组织样品：动物用含有20U/ml Heparin的生理盐水(0.9% NaCl containing 20U/ml Heparin)灌流清除血液后获取组织样品，按照每100mg组织加入500μl磷酸缓冲液(pH7.8)的比例，用玻璃匀浆器在4℃或冰浴匀浆，4℃ 4000r/min离心10min，取上清液(SOD粗提液)用于酶活性的测定。
③细胞样品：对于贴壁细胞，由于后续用于酶活性的测定，避免使用胰酶消化细胞。可以使用细胞刮或EDTA处理细胞并收集细胞，细胞用无菌的PBS或生理盐水洗涤1次，按照每106细胞加入300～500μl磷酸缓冲液(pH7.8)的比例，用玻璃匀浆器在4℃或冰浴匀浆，4℃ 10000r/min离心10min，取上清液(SOD粗提液)用于酶活性的测定。
④植物样品：准确称取植物材料(果肉或者去叶脉的叶片)0.4g，剪碎，置于4℃预冷的研钵或匀浆器中，加入预冷磷酸缓冲液(pH7.8) 1ml，低温研磨至匀浆后转移至离心管，用3ml磷酸缓冲液冲洗研钵或匀浆器并转入离心管，加总体积至4ml，4℃ 10000r/min离心20min，上清液为酶提取液，可用于SOD的检测。
⑤澄清液体样品可取原液直接测定，浑浊液体样品可经4℃ 4000r/min离心15min，再取上清液测定。
※注意：如果SOD酶活性较低，应相应减少提取液的总体积，以便提高SOD酶的浓度。提取液建议使用磷酸缓冲液(50mM pH7.8)，也可以根据需要使用蒸馏水或其他溶液。
⑥样品准备完毕后可以用BCA蛋白定量检测试剂盒测定蛋白浓度，通常10～20μg蛋白的细胞或组织匀浆液样品中的SOD平均活力约1个活力单位左右(不同细胞和组织的差异会比较大，该活力范围仅作为初步的参考)。每种样品准备20～100μg蛋白量通常已经足够用于后续检测，根据蛋白浓度和预计的蛋白使用量，用相关提取液适当稀释样品；例如小鼠肝脏组织10%匀浆液(组织和匀浆液的重量比为10%)上清，通常需要稀释10～100倍，准备好的样品如果当天测定，可以冰浴保存；如果当天不能完成测定，可以-20℃冻存，但建议尽量当天完成测定。
2、邻苯三酚自氧化速率测定：在25℃左右，于两个离心管中按下表依次加入各试剂。
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加入显色液后立即混匀倾入1cm比色皿内，在325nm波长下测定0s、30s、60s、90s、120s、150s、180s两个管的吸光值，计算线性范围内每分钟吸光度的增值(自氧化测定管-自氧化空白管)，即邻苯三酚的自氧化速率∆A325(min-1)，该试剂盒经测定∆A325(min-1)约为0.060，可通过调节邻苯三酚的加入量控制自氧化速率在每分钟0.060~0.07。
3、 样品SOD抑制邻苯三酚自氧化速率测定：按照下表依次加入相应成分，加入显色液后立即混匀倾入1cm比色皿内，在325nm波长下测定两个管的吸光值，使抑制邻苯三酚自氧化的速率约为1/2邻苯三酚的自氧化速率，即∆A´325(min-1)为0.030。
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计算：
SOD活力单位定义：25℃时，在1ml反应液中每分钟抑制邻苯三酚自氧化速率达50%时的酶量定义为1个活性单位(U)，即在325nm处为0.03OD/min为一个活力单位。若自氧化速率为35%~65%，通常可按比例计算，不在此范围内的数值应增减样液用量。
抑制率
= [ΔA₃₂₅(min⁻¹) − ΔA′₃₂₅(min⁻¹)] / ΔA₃₂₅(min⁻¹) × 100%
液体样品中总 SOD 活力(U/ml)
= 抑制率 / 50% × 1.8 × (1/V) × D
组织、细胞、植物等固体样品匀浆液中总 SOD 活力(U/g)
血液中总 SOD 活力(U/g·Hb)
= 血液中总 SOD 活力(U/ml) / Hb(g·Hb/ml) × 1000
式中：
ΔA₃₂₅(min⁻¹) = 邻苯三酚自氧化速率
ΔA′₃₂₅(min⁻¹) = 样品管抑制邻苯三酚自氧化速率
1.8 = 反应液总体积(ml)
V = 测定时样品所用体积(ml)
D = 提取液稀释倍数
VT = SOD 提取液总体积(ml)
m = 样品质量(g)
V₀ = 采血量(ml)
Hb = 血红蛋白含量(g·Hb/ml)
注意事项：
1、待测样品 - 70℃可保存 1 个月，需注意反复冻融会导致 SOD 部分失活。
2、细胞或组织等样品制备时不易采用含有 Triton X-100 等去垢剂的溶液。
3、抗氧化物会对本试剂盒的检测产生干扰，例如 0.1mM ascorbic acid、5mM GSH 以及维生素 C 都会使测定出来的吸光度显著升高，应设法除去或不添加相关成分。
4、对于植物样品，研磨处理应迅速，以免 SOD 酶活下降，尽量在冰浴条件下处理样品。
5、在邻苯三酚自氧化速率与酶活力测定过程中，记录时间应准确一致，以保证吸光度读数的准确性。
6、反应温度、pH 和邻苯三酚的浓度都对结果有影响，故应严格控制。
7、所有实验器材必须清洁干燥。8、SOD 对邻苯三酚自氧化速率的抑制率在 5min 内呈线性。
9、邻苯三酚自氧化速率以 0.06 为标准，可控制在 0.06~0.07 之间，可通过适当增减邻苯三酚的用量加以调节。
10、为了您的安全和健康，请穿实验服并戴一次性手套操作。
11、试剂开封后请尽快使用，以防影响后续实验效果。
Superoxide Dismutase (SOD) is a metalloenzyme that catalyzes the dismutation of superoxide anion into hydrogen peroxide (H₂O₂) and oxygen (O₂). It is an important antioxidant enzyme in organisms. Since superoxide radicals are unstable with an extremely short half-life, SOD activity is generally measured by indirect methods using various colorimetric reactions. Common chromogenic agents include NBT (Nitro Blue Tetrazolium), WST-1, and WST-8.
Total Superoxide Dismutase (SOD) Assay Kit (Pyrogallol Colorimetric Method), also known as the pyrogallol autoxidation method or pyrogallol autoxidation inhibition method, works on the principle that pyrogallol undergoes rapid autoxidation under alkaline conditions, releasing superoxide anion radicals and forming yellow intermediate products. In the initial stage of autoxidation, the accumulation of yellow products shows a linear relationship with time after a lag phase of 30–45 seconds, and these products have a strong absorption at 325 nm. When SOD is present, it catalyzes the reaction between superoxide anion radicals and hydrogen ions to form oxygen and hydrogen peroxide, thereby inhibiting the accumulation of intermediate products. Therefore, SOD activity can be determined based on its ability to inhibit pyrogallol autoxidation. This kit is for research use only and is not intended for clinical diagnosis or other purposes.
Kit Components:
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Materials Required but Not Provided:
Assay Procedure (For Reference Only):
1. Sample Preparation
① Plasma or red blood cell-containing samples:For plasma SOD activity assay: Separate serum or plasma from the test sample; avoid hemolysis. If red blood cells are present, centrifuge at 3000 r/min for 5 min at 4°C, transfer the supernatant to a new centrifuge tube, and dilute with an appropriate volume of normal saline before testing. If the value exceeds the detection range, dilute with phosphate buffer (pH 7.8) and retest.For erythrocyte SOD activity assay: Take a certain volume of fresh blood or heparin-anticoagulated blood, mix well, centrifuge at 3000 r/min for 5 min, discard the supernatant, lyse the pellet completely with ACK erythrocyte lysis buffer, centrifuge again at 3000 r/min for 5 min. The supernatant is the crude erythrocyte SOD extract.
② Tissue samples:Perfuse animals with normal saline containing 20 U/mL heparin (0.9% NaCl containing 20 U/mL Heparin) to remove blood before harvesting tissues. Homogenize the tissues with a glass homogenizer at 4°C or on ice in phosphate buffer (pH 7.8) at a ratio of 500 μL buffer per 100 mg tissue. Centrifuge the homogenate at 4000 r/min for 10 min at 4°C. Collect the supernatant (crude SOD extract) for enzyme activity assay.
③ Cell samples:For adherent cells, avoid trypsin digestion. Harvest cells using a cell scraper or EDTA treatment, wash once with sterile PBS or normal saline. Homogenize cells with a glass homogenizer at 4°C or on ice in phosphate buffer (pH 7.8) at a ratio of 300–500 μL buffer per 10⁶ cells. Centrifuge at 10000 r/min for 10 min at 4°C. Collect the supernatant (crude SOD extract) for enzyme activity assay.
④ Plant samples:Accurately weigh 0.4 g of plant material (pulp or leaf blades without veins), cut into small pieces, and place in a pre-chilled mortar or homogenizer at 4°C. Add 1 mL of pre-chilled phosphate buffer (pH 7.8), grind to a homogenate at low temperature, transfer to a centrifuge tube, rinse the mortar/homogenizer with 3 mL phosphate buffer and combine into the same tube to make a total volume of 4 mL. Centrifuge at 10000 r/min for 20 min at 4°C. The supernatant is the enzyme extract, ready for SOD assay.
⑤ Clear liquid samples can be tested directly. Turbid liquid samples should be centrifuged at 4000 r/min for 15 min at 4°C before collecting the supernatant for testing.
※ Note: If SOD activity is low, reduce the total volume of the extract accordingly to increase SOD concentration. It is recommended to use phosphate buffer (50 mM, pH 7.8) as the extractant; distilled water or other solutions may also be used as needed.
⑥ After sample preparation, determine the protein concentration using a BCA protein assay kit. Typically, 10–20 μg of protein from cell or tissue homogenate corresponds to approximately 1 unit of SOD activity (values vary widely between different cells and tissues; this range is only a preliminary reference). Prepare 20–100 μg of total protein per sample, which is usually sufficient for subsequent assays. Dilute the sample appropriately with the extractant based on protein concentration and expected usage. For example, the supernatant of a 10% mouse liver homogenate (10% w/v tissue to buffer ratio) is usually diluted 10–100 times. Prepared samples can be stored on ice if assayed the same day; if not assayed immediately, they can be frozen at -20°C, but it is recommended to complete the assay on the same day.
2. Determination of Pyrogallol Autoxidation Rate
At approximately 25°C, add reagents to two centrifuge tubes as follows:
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After adding the color developing solution, mix immediately and transfer to a 1 cm cuvette. Measure the absorbance of both tubes at 325 nm at 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s. Calculate the absorbance increase per minute within the linear range (Autoxidation Assay tube – Autoxidation Blank tube), i.e., the pyrogallol autoxidation rate ΔA₃₂₅(min⁻¹). The expected ΔA₃₂₅(min⁻¹) for this kit is approximately 0.060; adjust the volume of pyrogallol added to control the autoxidation rate between 0.060–0.070 per minute.
3. Determination of SOD-Mediated Inhibition of Pyrogallol Autoxidation
Add the corresponding components as follows. After adding the color developing solution, mix immediately and transfer to a 1 cm cuvette. Measure the absorbance of both tubes at 325 nm. The rate of pyrogallol autoxidation should be inhibited to approximately half the autoxidation rate, i.e., ΔA′₃₂₅(min⁻¹) should be 0.030.
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Calculations
Definition of SOD Activity Unit: At 25°C, the amount of enzyme that inhibits pyrogallol autoxidation by 50% per minute in 1 mL reaction solution is defined as 1 activity unit (U), corresponding to a change of 0.03 OD/min at 325 nm. If the inhibition rate is between 35%–65%, calculate proportionally; if outside this range, adjust the sample volume accordingly.
Inhibition Rate
= [ΔA₃₂₅(min⁻¹) − ΔA′₃₂₅(min⁻¹)] / ΔA₃₂₅(min⁻¹) × 100%
Total SOD Activity in Liquid Samples (U/mL)
= Inhibition Rate / 50% × 1.8 × (1/V) × D
Total SOD Activity in Homogenates of Tissues, Cells, and Plants (U/g)
= Inhibition Rate / 50% × 1.8 × (1/V) × D × (VT/m)
Total SOD Activity in Blood (U/mL)= Inhibition Rate / 50% × 1.8 × (1/V) × D × (VT/V₀)
Total SOD Activity in Blood (U/g·Hb)
= Total SOD Activity in Blood (U/mL) / Hb(g·Hb/ml) × 1000
Where:
ΔA₃₂₅(min⁻¹) = Pyrogallol autoxidation rate
ΔA′₃₂₅(min⁻¹) = Rate of pyrogallol autoxidation inhibited by the sample
1.8 = Total volume of the reaction solution (mL)
V = Volume of sample used in the assay (mL)
D = Dilution factor of the extract
VT = Total volume of the SOD extract (mL)
m = Sample mass (g)
V₀ = Volume of blood collected (mL)
Hb = Hemoglobin concentration (g·Hb/mL)
Notes:
| T1521754 | 组件 | 物理外观 | 50T | 储存 |
| T1521754A | SOD 检测缓冲液 | 液体 | 50 mL | 2-8℃. |
| T1521754B | 邻苯三酚显色液 | 液体 | 5 mL | 2-8℃. 避光. |
| T1521754C | 空白对照液 | 液体 | 5 mL | 2-8℃. |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838529 |
分析证书 | T1521754 |
| ZJ26F0535768 |
分析证书 | T1521754 |
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