BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
乙酸激酶（Acetokinase, ACK）主要存在于微生物中，催化乙酸和ATP生成乙酰磷酸和ADP，是细菌碳代谢和能量代谢的关键酶，尤其是在古细菌甲烷合成代谢中起着中枢作用。
测定原理：ACK催化乙酸钠和ATP生成乙酰磷酸和ADP，丙酮酸激酶催化ADP和PEP生成ATP 和丙酮酸，乳酸脱氢酶催化丙酮酸和NADH生成乳酸和NAD+，在340nm下测定NADH氧化生成NAD+速率，即可反映ACK 活性。
| Component | 50T | Storage |
| 提取液 | 80 mL | 2-8℃ |
| 试剂一 | 1EA | -20℃ |
| 试剂二 | 4EA | -20℃ |
| 试剂三 | 2EA | -20℃ |
| 试剂四 | 2EA | -20℃ |
试剂一：用前甩几下使试剂落入底部，再加8.4mL的蒸馏水溶解备用，用不完的试剂分装后-20℃保存。
试剂二：每支用前甩几下使试剂落入底部， 再加0.6mL 的蒸馏水溶解备用。用不完的试剂分装后-20℃保存，禁止反复冻融，三天内用完。
试剂三：临用前甩几下使粉剂落入底部，每支再加0.6mL蒸馏水充分溶解备用，可分装冻存，禁止反复冻融。
试剂四： 临用前甩几下使粉剂落入底部，每支再加0.6mL蒸馏水充分溶解备用，可分装冻存，禁止反复冻融。
自备仪器和用品
紫外分光光度计、1mL石英比色皿（光径1cm）、低温台式离心机、恒温培养箱、移液器、研钵、 冰和蒸馏水。
样本制备
1、组织样本：称取约0.1g组织，加入1mL提取液，进行冰浴匀浆。12000rpm，4℃离心10min，取上清，置冰上待测。
注：若增加样本量，可按照组织质量（g）：提取液体积(mL)为1：5~10的比例进行提取。
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500万细菌或细胞加入1mL提取液；冰浴超声波破碎细菌或细胞（冰浴，功率20％或200W，超声3s，间隔10s，重复30次）；12000rpm，4℃离心10min，取上清，置冰上待测。
注：若增加样本量，可按照细菌/细胞数量（10⁴个）：提取液（mL）为1:1000~5000比例进行提取。
测定步骤：
1、紫外分光光度计预热30min以上，调节波长至340nm，蒸馏水调零。
2、所有试剂可放在37℃水浴5-15min。
3、提取液和试剂一和二和三和四可按照400:160:40:20:20比例配成混合液（一枪加640μL 该混合液）（该混合液用多少配多少，现配现用）。
4、依次在1mL石英比色皿（光径1cm）中加入：
| 试剂名称（μL） | 测定管 |
| 提取液 | 400 |
| 试剂一 | 160 |
| 试剂二 | 40 |
| 试剂三 | 20 |
| 试剂四 | 20 |
| 混匀，37℃下，孵育5min | |
| 样本 | 60 |
混匀，10s时于340nm处读取吸光值A1， 10min后读取吸光值A2，ΔA=A1-A2。
注：
1、若ΔA的值在零附近，可以适当延长反应时间到20min或更长读取A2，改变后的反应时间需代入计算公式重新计算。或适当加大样本量V1（如增至100μL，则提取液相应减少），则改变后的加样体积V1和反应时间T需代入计算公式重新计算。
2、若起始值A1太大如超过2（如颜色较深的植物叶片，一般色素较高，则起始值相对会偏高），可以适当减少样本加样量V1，则改变后的加样体积V1需代入计算公式重新计算。或向待测样本中加少许活性炭混匀静置5min后12000rpm，4℃离心10min，上清液用于检测。
3、若ΔA大于0.6，可减少反应时间时间（如5min），则改变后的反应时间T代入计算公式重新计算。
4、若下降趋势不稳定，可以每隔10S读取一次吸光值，选取一段线性下降的时间段来参与计算，相对应的A值也代入计算公式重新计算。
ACK活性计算：
1、按样本蛋白浓度计算
酶活定义：每毫克组织蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。 ACK(nmol/min/mgprot)=[ΔA÷(ε×d)×V2×10⁹]÷(Cpr×V1÷V)÷T=187.6×ΔA÷Cpr
2、按样本鲜重计算
酶活定义：每克组织每分钟消耗1nmol NADH定义为一个酶活力单位。
ACK(nmol/min/g鲜重)=[ΔA÷(ε×d)×V2×10⁹]÷(W×V1÷V)÷T=187.6×ΔA÷W
3、按细菌/细胞密度计算
酶活定义：每1万个细菌/细胞每分钟消耗1nmol NADH定义为一个酶活力单位。 ACK(nmol/min/10⁴cell)=[ΔA÷(ε×d)×V2×10⁹]÷(500×V1÷V)÷T=0.38×ΔA
ε：NADH摩尔消光系数，6.22×10³L/mol/cm
d：光径，1cm
V：加入提取液体积，1mL
V1：加入样本体积，0.06mL
V2：反应体系总体积，7×10⁻⁴L
T：反应时间，10min
500：细菌或细胞总数，万
W：样本质量，g
Cpr：样本蛋白质浓度，mg/mL
注意事项
正式测定前务必取2-3个预期差异较大的样本做预测定。
Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.
Assay Principle
ACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.
| Component | 50T | Storage |
| Extraction Buffer | 80 mL | 2-8℃ |
| Reagent 1 | 1EA | -20℃ |
| Reagent 2 | 4EA | -20℃ |
| Reagent 3 | 2EA | -20℃ |
| Reagent 4 | 2EA | -20℃ |
Reagent Preparation
Reagent 1: Before use, centrifuge the vial to bring the powder to the bottom. Add 8.4 mL of distilled water to dissolve. Aliquot and store unused portions at -20°C.
Reagent 2: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.
Reagent 3: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.
Reagent 4: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.
Required Materials and Equipment (Not Provided)
UV spectrophotometer, 1 mL quartz cuvette (1 cm light path), refrigerated benchtop centrifuge, constant temperature incubator, pipettes, mortar and pestle, ice, and distilled water.
Sample Preparation
1.Tissue Samples: Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 12,000 rpm (approx. 13,000-15,000 g), 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Note: For larger samples, use an Extraction Buffer volume (mL) to tissue mass (g) ratio between 5:1 and 10:1.
2.Bacteria/Cell Samples: Collect cells by centrifugation and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million cells/bacteria. Disrupt the cells/bacteria by sonication on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 12,000 rpm, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Note: For larger samples, use an Extraction Buffer volume (mL) to cell count (10⁴ cells) ratio between 1:1000 and 1:5000.
Assay Procedure:
1.Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.
2.Pre-warm all reagents at 37°C for 5-15 minutes.
3.Optional Master Mix: A Master Mix can be prepared just before use by combining Extraction Buffer, Reagent 1, Reagent 2, Reagent 3, and Reagent 4 in a 400:160:40:20:20 ratio (e.g., for one assay: 400μL + 160μL + 40μL + 20μL + 20μL = 640μL). Pipette 640 μL of this Master Mix per cuvette. Prepare the Master Mix fresh for immediate use.
4.Pipette into a 1 mL quartz cuvette (1 cm light path) in the following order:
| Reagent | Volume (μL) |
| Extraction Buffer | 400 |
| Reagent 1 | 160 |
| Reagent 2 | 40 |
| Reagent 3 | 20 |
| Reagent 4 | 20 |
| Mix thoroughly and incubate at 37°C for 5 min. | |
| Sample | 60 |
Mix thoroughly immediately after adding the sample. Record the initial absorbance (A₁) at 340 nm at 10 seconds. Record the absorbance again (A₂) after exactly 10 minutes. Calculate ΔA = A₁ - A₂.
Notes & Troubleshooting:
1.If ΔA is close to zero, consider extending the reaction time (e.g., to 20 min) to read A₂. Use the new reaction time (T) in the calculation. Alternatively, increase the sample volume V₁ (e.g., to 100 μL, decrease Extraction Buffer accordingly) and use the new V₁ and T in the calculation.
2.If the initial absorbance A₁ is too high (e.g., >2, common in deeply pigmented plant samples), reduce the sample volume V₁ and use the new V₁ in the calculation. Alternatively, add a small amount of activated charcoal to the sample supernatant, mix, let stand for 5 min, centrifuge at 12,000 rpm, 4°C for 10 min, and use the clarified supernatant for assay.
3.If ΔA is greater than 0.6, reduce the reaction time (e.g., to 5 min) and use the new time (T) in the calculation.
4.If the decrease is not linear, read the absorbance every 10 seconds and select a linear segment for calculating ΔA. Use the corresponding time interval (T) for the calculation.
ACK Activity Calculation:
General Parameters:
ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cm
d (Cuvette light path) = 1 cm
V (Total extraction volume) = 1 mL
V₁ (Sample volume in reaction) = 0.06 mL (60 μL)
V₂ (Total reaction volume) = 0.0007 L (700 μL)
T (Reaction time) = 10 min
500 (Cell/Bacteria count in ten-thousands: 500 × 10⁴ = 5 million)
W (Sample mass, g)
Cpr (Sample protein concentration, mg/mL)
1. Based on Sample Protein Concentration:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.
Calculation:
ACK Activity (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (Cpr × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/mg prot) = 187.6 × ΔA ÷ Cpr
2. Based on Sample Fresh Weight:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh tissue.
Calculation:
ACK Activity (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/g fresh weight) = 187.6 × ΔA ÷ W
3. Based on Bacterial/Cell Density:
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells/bacteria.
Calculation (for 5 million cells in 1 ml extract):
ACK Activity (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/10⁴ cell) = 0.38 × ΔA
Precautions
Before formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.
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