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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。
葡萄糖激酶（GK，EC2.7.1.2）是己糖激酶家族中的一员，主要存在于成熟的肝细胞和胰岛细胞中。 在正常的生理条件下，GK的主要作用是监控血中葡萄糖水平。
检测原理：葡萄糖激酶 (GK) 磷酸化葡萄糖并产生6-磷酸葡萄糖，该产物进一步与6-磷酸葡萄糖脱氢酶和 NADP+偶联，在340nm测NADPH光吸收增加量，进而计算出该酶活性大小。
| Component | 100T | Storage |
| 提取液 | 120 mL | 2-8℃ |
| 试剂一 | 20 mL | 2-8℃ |
| 试剂二 | 1EA | -20℃ |
| 试剂三 | 1EA | 2-8℃ |
试剂二（粉剂1支）：
1、临用前 8000g 4°C离心 2mim 使试剂落入管底（可手动甩一甩）；
2、加入1.1mL蒸馏水溶解备用；
3、保存周期与试剂盒有效期相同。
试剂三（粉剂1支）：
1、开盖前注意使粉剂落入底部（可手动甩一甩）；
2、加入18mL试剂一溶解备用；
3、保存周期与试剂盒有效期相同。
自备仪器和用品
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）、 96 孔板、离心管、酶标仪、蒸馏水（去离子水、超纯水均可）。
实验流程
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
1.1 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，可以按照组织质量（g）：提取液体积(mL)为1：5~10的比例提取。
1.2 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，可按照细菌或细胞数量（10⁴个）：提取液体积（mL）为500~1000：1的比例进行提取。
1.3 液体样本
直接检测。若浑浊，离心后取上清检测。
2、检测步骤
2.1 酶标仪预热30min以上，调节波长至340nm。
2.2 配置好的试剂二和三在25℃预热5min至室温；
2.3 在96孔板中依次加入：
| 试剂 (μL) | 测定管 |
| 样本 | 20 |
| 试剂二 | 10 |
| 试剂三 | 170 |
混匀，1min时于340nm处读取吸光值A1， 21min（即20min后）读取A2，ΔA=A2-A1。
注：
若ΔA的值在零附近，可以适当延长反应时间到30min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，则改变后的加样体积V1需代入计算公式重新计算。
若上升趋势不稳定，可以每隔10S读取一次吸光值，选取一段线性上升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。
3、结果计算
3.1 按样本蛋白浓度计算
单位定义：每毫克组织蛋白每分钟生成1nmol的NADPH定义为一个酶活力单位。
葡萄糖激酶 (GK)（nmol/min/mg prot）=[ΔA÷（ε×d）×V2×10⁹]÷(V1×Cpr) ÷T=160.77×ΔA÷Cpr
3.2 按样本鲜重计算
单位定义：每g组织每分钟生成1nmol的NADPH定义为一个酶活力单位。
葡萄糖激酶 (GK)（nmol/min/g 鲜重）=[ΔA÷（ε×d）×V2×10⁹]÷(W×V1÷V) ÷T=160.77×ΔA÷W
3.3 按细菌或细胞密度计算
单位定义：每1万个细菌或细胞每分钟生成1nmol的NADPH定义为一个酶活力单位。
葡萄糖激酶(GK) (nmol/min/10⁴ cell)=[ΔA÷(ε×d)×V2×10⁹]÷(500×V1÷V)÷T=0.32×ΔA
3.4 按液体体积计算
单位定义：每毫升液体在每分钟生成1nmol的NADPH定义为一个酶活力单位。
葡萄糖激酶 (GK)(nmol/min/mL)=[ΔA×V2÷(ε×d)×10⁹]÷V1÷T=160.77×ΔA
参数说明
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔板光径，0.5cm；
V：加入提取液体积，1mL；
V1：加入样本体积，0.02mL；
V2：反应体系总体积，0.2mL=2.0×10⁻⁴ L；
T：反应时间，20min；
W：样本质量，g；
500：细菌或细胞数量，500万；
Cpr：样本蛋白质浓度，mg/mL；推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
注意事项
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。
Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels.
Detection Principle: Glucokinase (GK) phosphorylates glucose to produce glucose-6-phosphate. This product is further coupled with glucose-6-phosphate dehydrogenase and NADP⁺. The increase in NADPH absorbance at 340 nm is measured, allowing for the calculation of the enzyme's activity.
| Component | 100T | Storage |
| Extraction Buffer | 120 mL | 2-8℃ |
| Reagent 1 | 20 mL | 2-8℃ |
| Reagent 2 | 1EA | -20℃ |
| Reagent 3 | 1EA | 2-8℃ |
Reagent 2 (Powder, 1 vial) Preparation:
Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube (can be flicked manually).
Add 1.1 mL of distilled water to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
Reagent 3 (Powder, 1 vial) Preparation:
Before opening, ensure the powder is at the bottom (can be flicked manually).
Add 18 mL of Reagent 1 to dissolve. Use after preparation.
The prepared solution can be stored for the duration of the kit's validity period.
User-Prepared Instruments and Materials
Mortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 96-well plate, Centrifuge tubes, Microplate reader, Distilled water (Deionized water or Ultrapure water are also acceptable).
Experimental Procedure
It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10.
1.2 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 20% or 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1.
1.3 Liquid Samples
Assay directly. If turbid, centrifuge and use the supernatant for assay.
2. Assay Steps
2.1 Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.
2.2 Pre-warm the prepared Reagent 2 and Reagent 3 at 25°C for 5 minutes to reach room temperature.
2.3 Add reagents sequentially to a 96-well plate:
| Reagent | Test Well (μL) |
| Sample | 20 |
| Reagent 2 | 10 |
| Reagent 3 | 170 |
Mix thoroughly. Read the absorbance at 340 nm at 1 minute (A₁) and again at 21 minutes (A₂, i.e., after 20 minutes of reaction). Calculate ΔA = A₂ - A₁.
Note:
If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A₂. If the reaction time is changed, the new time (T) must be substituted into the calculation formula. Alternatively, the sample volume can be increased; the new sample volume (V₁) must then be substituted into the calculation formula.
If the increase trend is unstable, read the absorbance every 10 seconds and select a linearly increasing time period for calculation. The corresponding A values for this period should be used to calculate ΔA and substituted into the formula.
3. Calculation of Results
3.1 Based on Sample Protein Concentration
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mg of tissue protein.
Derived Formula: GK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (V₁ × Cpr) ÷ T
Simplified Formula: GK (nmol/min/mg prot) = 160.77 × ΔA ÷ Cpr
3.2 Based on Sample Fresh Weight
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per gram of tissue.
Derived Formula: GK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ ÷ V) ÷ T
Simplified Formula: GK (nmol/min/g fresh weight) = 160.77 × ΔA ÷ W
3.3 Based on Bacterial or Cell Density
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per 10⁴ bacteria or cells.
Derived Formula: GK (nmol/min/10⁴ cells) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ ÷ V) ÷ T
Simplified Formula: GK (nmol/min/10⁴ cells) = 0.32 × ΔA
3.4 Based on Liquid Volume
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mL of liquid.
Derived Formula: GK (nmol/min/mL) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ V₁ ÷ T
Simplified Formula: GK (nmol/min/mL) = 160.77 × ΔA
Parameter Definitions:
ε: Molar extinction coefficient of NADPH (6.22 × 10³ L/mol/cm)
d: Light path length for the 96-well plate (0.5 cm)
V: Volume of Extraction Buffer added (1 mL)
V₁: Volume of sample added to the reaction (0.02 mL)
V₂: Total volume of the reaction system (0.2 mL = 2.0 × 10⁻⁴ L)
T: Reaction time (20 minutes)
W: Sample weight (g)
500: Total number of bacteria or cells (5 million)
Cpr: Sample protein concentration (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
Precautions
It is strongly recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.
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