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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。
精氨酸脱羧酶 (ADC，EC 4.1.1.19) 是多数植物体内催化游离态多胺合成的关键酶，与植物逆境生理有一定关系。
精氨酸脱羧酶 (ADC) 催化底物精氨酸生成产物胍丁胺，通过衍生剂使产物胍丁胺衍生化，该衍生物在 254nm 处有最大吸收峰。通过检测 254nm 处吸光值变化得出 ADC 酶活大小。
组分与储存
1. 若重新做标曲，则用到该试剂； 2. 按照说明书中标曲制作步骤进行配制； 3. 溶解后的标品一周内用完。 |
自备仪器和试剂
⁕ 紫外分光光度计 (能测254nm处的吸光度)
⁕ 研钵 (匀浆机)、冰盒 (制冰机)、台式离心机、可调式移液枪、水浴锅 (烘箱、培养箱、金属浴)、1mL石英比色皿、离心管、氮吹仪、涡旋振荡仪 (超声仪)
⁕ 乙醇、乙酸乙酯、甲醇、蒸馏水 (去离子水、超纯水均可)
指标测定
建议先选取 1-3 个差异大的样本 (例如不同类型或分组) 进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1. 样本提取：
① 称取约 0.2g 组织，加入 1mL 经预冷的 95%乙醇冰浴匀浆，4℃放置 10min；12000rpm，4℃离心 5min；弃上清，留沉淀，向沉淀中加入经预冷的 80%乙醇混匀，4℃放置 10min；12000rpm，4℃离心 5min；弃上清，留沉淀。再向沉淀中加入 1mL 经预冷提取液，涡旋混匀，4℃放置 10min；12000rpm，4℃离心 5min；留上清，弃沉淀。上清液置冰上待测。
② 细菌/细胞样本：先收集细菌或细胞到离心管内，离心后弃上清；取约 500 万细菌或细胞加入1mL 经预冷的 95%乙醇冰浴匀浆，4℃放置 10min；12000rpm，4℃离心 5min；弃上清，留沉淀，向沉淀中加入经预冷的 80%乙醇混匀，4℃放置 10min；12000rpm，4℃离心 5min；弃上清，留沉淀。再向沉淀中加入 1mL 经预冷提取液，涡旋混匀，4℃放置 10min；超声波破碎细菌或细胞 (冰浴，功率 200W，超声 3s，间隔 10s，重复 30 次)；12000rpm 4℃离心 10min，取上清，置冰上待测。上清液置冰上待测。
【注】：若增加样本量，可按照细菌/细胞数量 (10⁴)：提取液 (mL) 为 500~1000：1 的比例进行提取。
③ 液体样本：直接检测；若浑浊，离心后取上清检测。
2. 检测步骤：
① 紫外分光光度计调至 254nm (等待仪器过自检程序亦可)。试剂一和二可于 37℃孵育 5min。在 EP管中依次加入：
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② 在 EP 管中依次加入：
1. 试剂五添加后，快速手动或涡旋仪混匀20秒，于40℃条件下孵育50min (期间振荡混匀3-5次，每次30秒)。 2. 全部试剂添加完成后，上下颠倒混匀约1分钟后 (试剂最好上下充分混匀几次)，12000rpm 室温离心3min 使试剂上下分层，取出 0.5mL上层液体至EP管中，接着用氮吹仪吹干，最后向沉淀中加入750μL甲醇使沉淀完全溶解 (可用涡旋振荡仪或超声仪)，最后取出全部澄清液体至1mL石英比色皿 (光径1cm) 中于254nm处读取吸光值A，ΔA=A测定-A对照 |
【注】1. 若 A>1.8，可对最后一步甲醇溶解液体再用甲醇稀释后测定，则稀释倍数 D 带入公式计算。
2. ΔA<0.01，则可加大样本取样 W；或增加加样体积 V₁ (由 150µL 增至 300µL 或更多，则试剂一相应减少)；或延长孵育时间 T (由 1 小时增至 2 小时)，则改变后的 W 和 V₁ 和 T 需带入公式重新计算。
结果计算
1. 标准曲线：y = 1.6694x - 0.0091；x 是标准品摩尔浓度 (µmol/mL)，y 是ΔA。
1. 按样本质量计算：
酶活定义：每克组织每小时催化 1µmol 精氨酸生成胍丁胺定义为一个酶活单位。
ADC (µmol/h/g 鲜重)=[(ΔA+0.0091)÷1.6694×0.6)]÷(W×V₁÷V)÷T×D=2.396×(ΔA+0.0091)÷W×D
2、按样本蛋白浓度计算：
酶活定义：每毫克样本蛋白每小时催化 1µmol 精氨酸生成胍丁胺定义为一个酶活单位。
ADC (µmol/h/mg prot)=[(ΔA+0.0091)÷1.6694×0.6)]÷(Cpr×V₁÷V)÷T×D=2.396×(ΔA+0.0091)÷Cpr×D
3、按液体体积计算：
酶活定义：每毫升液体样本每小时催化 1µmol 精氨酸生成胍丁胺定义为一个酶活单位。
ADC (µmol/h/mL)==[(ΔA+0.0091)÷1.6694×0.6)]÷V₁÷T×D=2.396×(ΔA+0.0091)×D
4.按细菌或细胞数量计算：
酶活定义：每10⁴个细胞/细菌每小时催化 1µmol 精氨酸生成胍丁胺定义为一个酶活单位。
ADC (µmol/h/10⁴ cell)=[(ΔA+0.0091)÷1.6694×0.6)]÷(500×V₁÷V)÷T×D=2.396×(ΔA+0.0091)÷500×D
V---加入提取液体积，1mL；
V₁---样本加入体积，0.15mL；
W---样本质量，g；
T---反应时间，1 小时；
D---稀释倍数，未稀释即为 1；
0.6---第①步中反应总体积，mL；
标准品分子量---228.27；
500---细菌或细胞总数，万；
Cpr---样本蛋白浓度，mg/mL；建议使用本公司的 BCA蛋白含量检测试剂盒 (R1491648)。
附：标准曲线制作过程：
1. 标曲为非必做实验，用户可根据实验需求制作标曲，亦可直接采用说明书计算公式进行结果计算。
2. 标准品母液浓度为 2mg/mL。将母液用甲醇稀释成六个浓度梯度的标准品，例如：0，0.02，0.04，0.06，0.08，0.1 mg/mL。也可根据实际样本调整标准品浓度。
3. 标品稀释参照表如下：
吸取标准品母液 100μL，加入 1.9ml 甲醇，混匀得到 0.1mg/mL 的标品稀释液待用。
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4. 依据②步测定管的加样表操作，根据结果，以各浓度吸光值减去 0 浓度 (空白管) 吸光值，过 0 点制作标准曲线。
1. 试剂五添加后，快速手动或涡旋仪混匀20秒，于40℃条件下孵育50min (期间振荡混匀3-5次，每次30秒)。 2. 全部试剂添加完成后，上下颠倒混匀约1分钟后 (试剂最好上下充分混匀几次)，12000rpm 室温离心3min 使试剂上下分层，取出 0.5mL上层液体至EP管中，接着用氮吹仪吹干，最后向沉淀中加入400μL甲醇使沉淀完全溶解 (可用涡旋振荡仪或超声仪)，最后取出全部澄清液体至1mL石英比色皿 (光径1cm) 中于254nm处读取吸光值A，ΔA=A标准-A空白 |
Arginine decarboxylase (ADC, EC 4.1.1.19) is a key enzyme catalyzing the biosynthesis of free polyamines in most plants and is closely related to plant stress physiology.
Arginine decarboxylase (ADC) catalyzes the substrate arginine to produce agmatine. The product agmatine is derivatized with a derivatizing agent, and the resulting derivative exhibits a maximum absorption peak at 254 nm. The ADC enzyme activity is determined by measuring the change in absorbance at 254 nm.
Contents & Storage
1. This reagent shall be used when re‑establishing the standard curve. 2. Prepare the solution following the standard‑curve preparation procedure described in the instruction manual. 3. Use up the dissolved standard within one week. |
Self‑supplied instruments and reagents
⁕ UV‑visible spectrophotometer (capable of measuring absorbance at 254 nm)
⁕ Mortar (homogenizer), ice box (ice maker), benchtop centrifuge, adjustable pipettors, water‑bath heater (oven, incubator, metal bath), 1 mL quartz cuvette, centrifuge tubes, nitrogen evaporator, vortex mixer (ultrasonic processor)
⁕ Ethanol, ethyl acetate, methanol, distilled water (deionized water or ultrapure water is acceptable)
Index determination
It is recommended to select 1‑3 samples with significant differences (e.g., different types or groups) for a pilot experiment to get familiar with the operating procedure. Determine or adjust sample concentration based on pilot‑test results to avoid unnecessary waste of samples and reagents!
1. Sample extraction
① Weigh approximately 0.2 g of tissue, add 1 mL pre‑cooled 95% ethanol, homogenize in an ice bath, and incubate at 4 °C for 10 min. Centrifuge at 12000 rpm, 4 °C for 5 min. Discard the supernatant and retain the pellet. Add pre‑cooled 80% ethanol to the pellet and mix well, incubate at 4 °C for 10 min. Centrifuge at 12000 rpm, 4 °C for 5 min. Discard the supernatant and retain the pellet. Add 1 mL pre‑cooled Extract solution to the pellet, vortex thoroughly, and incubate at 4 °C for 10 min. Centrifuge at 12000 rpm, 4 °C for 5 min. Collect the supernatant and discard the pellet. Keep the supernatant on ice for subsequent measurement.
② Bacterial / cell samples: Collect bacteria or cells into centrifuge tubes and discard supernatant after centrifugation. Take approximately 5 × 10⁶ bacteria or cells, add 1 mL pre‑cooled 95% ethanol, homogenize in an ice bath, and incubate at 4 °C for 10 min. Centrifuge at 12000 rpm, 4 °C for 5 min. Discard the supernatant and retain the pellet. Add pre‑cooled 80% ethanol to the pellet and mix well, incubate at 4 °C for 10 min. Centrifuge at 12000 rpm, 4 °C for 5 min. Discard the supernatant and retain the pellet. Add 1 mL pre‑cooled Extract solution to the pellet and vortex thoroughly, incubate at 4 °C for 10 min. Lyse bacteria or cells by sonication (ice‑bath condition, power 200 W, sonicate for 3 s, interval 10 s, repeat 30 cycles). Centrifuge at 12000 rpm, 4 °C for 10 min. Collect the supernatant and keep it on ice for measurement.
Note: If increasing sample loading, perform extraction following the ratio: bacterial/cell count (10⁴) : Extract solution (mL) = 500‑1000 : 1.
③ Liquid samples: Measure directly. If turbid, collect supernatant after centrifugation for detection.
2. Assay procedures
① Set the UV spectrophotometer to 254 nm (allow the instrument to complete self‑check). Reagent 1 and Reagent 2 can be pre‑incubated at 37 °C for 5 min. Add the following components sequentially into an EP tube:
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② Add the following components sequentially into the EP tube:
1. After adding Reagent 5, mix rapidly by hand or with a vortex mixer for 20 s, then incubate at 40 °C for 50 min (agitate‑mix 3‑5 times during incubation, 30 s each time). 2. After all reagents are added, invert to mix thoroughly for about 1 min (perform several complete inversion‑mixing cycles for sufficient mixing). Centrifuge at 12000 rpm for 3 min at room temperature to achieve phase separation. Transfer 0.5 mL of the upper‑phase liquid to a new EP tube, and dry the sample using a nitrogen evaporator. Re‑dissolve the residue completely with 750 μL methanol (vortex mixer or ultrasonic processor is acceptable). Transfer all of the clear solution into a 1 mL quartz cuvette (1 cm optical path), and record the absorbance value A at 254 nm. ΔA = Aassay − Acontrol. |
【Notes】
1. If A > 1.8, dilute the final methanol‑reconstituted solution with methanol prior to measurement. Include the dilution factor D in the calculation formula.
2. If ΔA < 0.01, increase sample weight W; or increase sample loading volume V₁ (increase from 150 µL to 300 µL or higher, with a corresponding reduction in Reagent 1 volume); or extend incubation time T (increase from 1 h to 2 h). Substitute the revised W, V₁ and T into the formula for recalculation.
Result calculation
1. Standard curve: y = 1.6694x - 0.0091; where x is the molar concentration of standard (µmol/mL), and y is ΔA.
1. Calculation based on sample mass
Enzyme activity definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol agmatine from arginine per gram of tissue per hour.
ADC (µmol/h/g fresh weight) = [(ΔA+0.0091)÷1.6694×0.6]÷(W×V₁÷V)÷T×D = 2.396×(ΔA+0.0091)÷W×D
2. Calculation based on sample protein concentration
Enzyme activity definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol agmatine from arginine per milligram of sample protein per hour.
ADC (µmol/h/mg prot) = [(ΔA+0.0091)÷1.6694×0.6]÷(Cpr×V₁÷V)÷T×D = 2.396×(ΔA+0.0091)÷Cpr×D
3. Calculation based on liquid‑sample volume
Enzyme activity definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol agmatine from arginine per milliliter of liquid sample per hour.
ADC (µmol/h/mL) = [(ΔA+0.0091)÷1.6694×0.6]÷V₁÷T×D = 2.396×(ΔA+0.0091)×D
4. Calculation based on bacterial or cell count
Enzyme activity definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol agmatine from arginine per 10⁴ cells/bacteria per hour.
ADC (µmol/h/10⁴ cell) = [(ΔA+0.0091)÷1.6694×0.6]÷(500×V₁÷V)÷T×D = 2.396×(ΔA+0.0091)÷500×D
V: Volume of Extract solution added, 1 mL
V₁: Volume of sample loaded, 0.15 mL
W: Sample mass, g
T: Reaction time, 1 h
D: Dilution factor; set to 1 if no dilution is performed
0.6: Total reaction volume in step ①, mL
Molecular weight of standard: 228.27
500: Total number of bacteria or cells (×10⁴)
Cpr: Protein concentration of sample, mg/mL; It is recommended to use our BCA Protein Assay Kit (R1491648).
Appendix: Standard curve preparation procedure
1. The standard‑curve assay is optional. Users may generate a standard curve according to experimental requirements, or directly apply the calculation formula provided in this manual for result computation.
2. The stock concentration of the standard is 2 mg/mL. Dilute the stock solution with methanol to prepare six standard concentrations, e.g., 0, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL. Standard concentrations can also be adjusted according to actual sample conditions.
3. Refer to the table below for standard dilution:
Pipette 100 μL of standard stock solution and add 1.9 mL methanol. Mix thoroughly to obtain 0.1 mg/mL diluted standard solution for subsequent use.
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4. Perform the assay following the pipetting scheme for assay tubes in Step ②. Subtract the absorbance value of the zero‑concentration (blank tube) from the absorbance of each standard concentration, and generate a zero‑intercept standard curve with the obtained data.
1. After adding Reagent Ⅴ, mix rapidly by hand or with a vortex mixer for 20 s, then incubate at 40 °C for 50 min (agitate‑mix 3‑5 times during incubation, 30 s each time). 2. After all reagents are added, invert to mix thoroughly for about 1 min (perform several complete inversion‑mixing cycles for sufficient mixing). Centrifuge at 12000 rpm for 3 min at room temperature to achieve phase separation. Transfer 0.5 mL of the upper‑phase liquid to a new EP tube, and dry the sample using a nitrogen evaporator. Re‑dissolve the residue completely with 400 μL methanol (vortex mixer or ultrasonic processor is acceptable). Transfer all of the clear solution into a 1 mL quartz cuvette (1 cm optical path), and record the absorbance value A at 254 nm. ΔA = Astandard − Ablank. |
| 组分货号 | 组分名称 | 物理外观 | 24T | 储存 |
| A1522219A | 提取液 | 液体 | 30 mL | 2-8℃. |
| A1522219B | 试剂一 | 液体 | 15 mL | 2-8°C. |
| A1522219C | 试剂二 | 液体 | 1.5 mL | 2-8°C. |
| A1522219D | 试剂三 | 液体 | 5 mL | 2-8℃. |
| A1522219E | 试剂四 | 液体 | 15 mL | 2-8℃. |
| A1522219F | Reagent 5 | 液体 | 0.3 mL | 2-8°C. 避光 |
| A1522219G | 试剂六 | 液体 | 30 mL | 2-8℃. |
| A1522219H | 标准品 | 液体 | 1 mL | 2-8°C. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939547 |
分析证书 | A1522219 |
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