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己糖激酶（Hexokinase, HK, EC2.7.1.1）广泛存在于动物、植物、微生物和培养细胞中，是葡萄糖分解过程中的第一个关键酶，催化葡萄糖转为6-磷酸葡萄糖，6磷酸葡萄糖是糖酵解和磷酸戊糖途径的交叉点。
检测原理：HK催化葡萄糖合成6-磷酸葡萄糖，6-磷酸葡萄糖脱氢酶进一步催化6-磷酸葡萄糖脱氢生成NADPH，NADPH在340nm有特征吸收峰。
| Component | 48T | 96T | Storage |
| Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| Reagent Ⅰ | 14 mL | 28 mL | 2-8℃ |
| Reagent Ⅱ | 1EA | 1EA | -20℃. Store in the dark. |
| Reagent Ⅲ | 1EA | 1EA | -20℃. Store in the dark. |
注：
请在实验前检查各组分的量。
各组分在规格基础上额外提供10%的量用于进行标准曲线制作或预实验。
自备仪器和用品
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测340nm处的吸光度 |
| 耗材 | 96孔UV板 | 紫外透明板 |
| 试剂 | 生理盐水 | 样本清洗用 |
| 其他 | 匀浆器（组织样本）、恒温箱、冰盒、低温离心机、 可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
使用说明
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Reagent Ⅰ | 即用型；使用前平衡到室温 | 4℃保存 |
| Working Reagent Ⅱ | 临用前配制； 48T加入10.8 mL ReagentⅠ溶解 96T加入21.6 mL ReagentⅠ溶解 | 实验过程中避光冰上放置；用不完的试剂-20℃分装避光保存一个月，禁止反复冻融 |
| Working Reagent Ⅲ | 临用前配制； 48T的Reagent Ⅲ加入0.5mL ReagentⅠ溶解 96T的Reagent Ⅲ加入1mL ReagentⅠ溶解； 根据用量将溶解的Reagent Ⅲ用ReagentⅠ稀释10倍为Working Reagent Ⅲ | 实验过程中避光冰上放置；用不完的试剂-20℃分装避光保存一个月，禁止反复冻融 |
2、样本制备
注意：推荐使用新鲜样本，样本经过冻存后HK活性会显著降低。
2.1 动植物组织
称取约0.1g组织样本，加入1mL Extraction Buffer，冰浴匀浆，8000g，4℃离心10min， 取上清置冰上待测。
2.2 细胞、细菌或真菌样本
收集5×10⁶个细胞、细菌或真菌，用预冷的生理盐水清洗细胞、细菌或真菌，800 g离心2min，弃上清。加入1mL Extraction Buffer进行冰浴超声破碎。超声波破碎5min（功率20%或 200 W，超声3s，间隔7s，重复30次），4℃，8000g离心10min，取上清置于冰上待测。
2.3 血清（浆）
直接测定。
3、实验步骤
3.1 酶标仪准备：预热30min以上，调节波长到340nm。
3.2 工作液的配制：临用前配制；每孔需要190μL工作液，为避免损失，可多配制2孔用量。按照190µL每单孔体系配制：分别吸取180μL Working Reagent Ⅱ和10μL Working Reagent Ⅲ混匀。工作液需现配现用，测定前在37℃孵育10min。
3.3 检测体系配制：在96孔UV板中按照如下方式操作
| 试剂 | 测定孔（μL） |
| 样本 | 10 |
| 工作液 | 190 |
3.4 吸光度检测：加入工作液后，立即混匀测定340nm处第10s时的吸光值A1和37℃准确孵育10min后第10min10s时的吸光值A2。
4、结果计算
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
4.1 数据处理
计算ΔA=A2-A1。
4.2 样本HK活性计算
① 按样本质量计算
酶活单位定义：每克样品每分钟生成1nmol NADPH定义为1个酶活单位。
HK (U/g)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 ×W)÷T×n=643.09×ΔA÷W×n
② 按细胞、细菌或真菌数量计算
酶活单位定义：每104个细胞、细菌或真菌每分钟生成1nmol NADPH定义为1个酶活单位。 HK(U/104)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 ×500)÷T×n=643.09×ΔA÷500×n
③ 按液体体积计算
酶活单位定义：每毫升液体每分钟生成1nmol NADPH定义为1个酶活单位。
HK(U/mL)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 )÷T×n=643.09×ΔA×n
④ 按蛋白浓度计算
酶活单位定义：每毫克蛋白每分钟生成1nmol NADPH定义为1个酶活单位。
HK(U/mg)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ×Cpr)÷T×n=643.09×ΔA÷Cpr×n
ε：NADPH摩尔消光系数，6.22×10³L/mol/cm
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。
3、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4、生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
5、本产品仅供科学研究使用，不适用于临床诊断。
相关问题及解答
1、检测得到的样本ΔA过高或者过低怎么办？
若ΔA>0.5，则说明样本HK活力太高，可用Extraction Buffer稀释成适当浓度匀浆上清液（计算公式中乘以相应稀释倍数），或缩短反应时间至5min。
若ΔA<0.005，则说明样本HK活力偏低，此时需加大样本量（此时工作液量不变，计算时变量代入计算公式）或是延长反应时间（如15min至30min）。
2、检测量较大时是否可以同时检测多个样本？
反应初始速度较快，不建议大量样本同时检测。如无多通道移液器，最好两个人同时做此实验，一个人比色，一个人计时，以保证实验结果的准确性。
Introduction
Hexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the pentose phosphate pathway.
Assay Principle
HK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.
| Component | 48T | 96T | Storage |
| Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| Reagent Ⅰ | 14 mL | 28 mL | 2-8℃ |
| Reagent Ⅱ | 1EA | 1EA | -20℃. Store in the dark. |
| Reagent Ⅲ | 1EA | 1EA | -20℃. Store in the dark. |
Note: Please check the quantity of all components before starting the experiment. An additional 10% of each component is provided for standard curve preparation or pilot experiments.
Required Materials and Equipment (Not Provided)
| Type | Name | Notes |
| Instrument | Microplate Reader | Must be capable of measuring absorbance at 340 nm |
| Consumables | 96-well UV Plate | UV-transparent plate |
| Reagents | Physiological Saline | For sample washing |
| Other | Homogenizer (for tissue samples), Incubator, Ice box, Refrigerated centrifuge, Adjustable pipettes and tips | Using a multichannel pipette is recommended for high-throughput experiments to improve efficiency |
Instructions for Use
1. Reagent Preparation
| 试剂名称 Reagent Name | Preparation | Notes |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature (RT) before use. | Store at 4°C |
| Reagent Ⅰ | Ready-to-use; equilibrate to room temperature (RT) before use. | Store at 4°C |
| Working Reagent Ⅱ | Prepare immediately before use: • For 48T: Dissolve contents of Reagent Ⅱ in 10.8 mL Reagent Ⅰ • For 96T: Dissolve contents of Reagent Ⅱ in 21.6 mL Reagent Ⅰ | Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles. |
| Working Reagent Ⅲ | Prepare immediately before use: 1. Dissolve Reagent Ⅲ: • For 48T: in 0.5 mL Reagent Ⅰ • For 96T: in 1 mL Reagent Ⅰ 2. Dilute the dissolved Reagent Ⅲ 10-fold with Reagent Ⅰ to make the Working Reagent Ⅲ. | Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles. |
2. Sample Preparation
Note: The use of fresh samples is highly recommended. HK activity decreases significantly upon sample freezing.
2.1 Animal/Plant Tissues
Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
2.2 Cells, Bacteria, or Fungi
Collect 5×10⁶ cells/bacteria/fungi. Wash with pre-cooled physiological saline and centrifuge at 800 g for 2 min. Discard the supernatant. Add 1 mL of Extraction Buffer and disrupt the cells by sonication on ice (5 min total, 20% power or 200 W, pulse 3s on/7s off, repeat 30 times). Centrifuge the lysate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
2.3 Serum (or Plasma)
Assay directly.
3. Assay Procedure
3.1 Microplate Reader Preparation: Preheat for at least 30 min. Set the wavelength to 340 nm.
3.2 Working Solution Preparation: Prepare immediately before use. Each well requires 190 µL of Working Solution. It is recommended to prepare enough for 2 extra wells to account for pipetting loss.
For a single well: Mix 180 µL of Working Reagent Ⅱ with 10 µL of Working Reagent Ⅲ.
The Working Solution must be prepared fresh. Incubate it at 37°C for 10 min before the assay.
3.3 Assay Setup: Pipette into wells of the 96-well UV plate as follows:
| Reagent | Test Well (µL) |
| Sample | 10 |
| Working Solution | 190 |
3.4 Absorbance Measurement: Immediately after adding the Working Solution, mix thoroughly and measure the absorbance at 340 nm at 10 seconds (A₁) and again after exactly 10 minutes of incubation at 37°C (at 10 min 10 sec, A₂).
4. Result Calculation
The following derived and simplified calculation formulas are provided and are equivalent.
4.1 Data Processing
Calculate ΔA = A₂ - A₁.
4.2 Sample HK Activity Calculation
① Based on Sample Mass (U/g)
Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of sample.
Calculation:
HK (U/g) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ T × n
Simplified Formula: HK (U/g) = 643.09 × ΔA ÷ W × n
② Based on Cell/Bacteria/Fungi Count (U/10⁴)
Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells/bacteria/fungi.
Calculation:
HK (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × 500) ÷ T × n
Simplified Formula: HK (U/10⁴) = 643.09 × ΔA ÷ 500 × n
③ Based on Liquid Volume (U/mL)
Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milliliter of liquid.
Calculation:
HK (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T × n
Simplified Formula: HK (U/mL) = 643.09 × ΔA × n
④ Based on Protein Concentration (U/mg prot)
Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milligram of protein.
Calculation:
HK (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T × n
Simplified Formula: HK (U/mg prot) = 643.09 × ΔA ÷ Cpr × n
Parameter Description:
ε: NADPH molar extinction coefficient = 6.22 × 10³ L/mol/cm
d: Light path of the 96-well UV plate = 0.5 cm
10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)
Vₜₒₜₐₗ: Total reaction volume = 200 μL = 2 × 10⁻⁴ L
Vₛₐₘₚₗₑ: Volume of supernatant added to the reaction = 10 μL = 1 × 10⁻⁵ L
Vₛₐₘₚₗₑₜₒₜₐₗ: Volume of Extraction Buffer added = 1 mL
Cpr: Sample protein concentration (mg/mL)
W: Sample mass (g)
T: Reaction time = 10 min
500: Cell/Bacteria/Fungi count = 5 × 10⁶, expressed in units of 10⁴
n: Sample dilution factor
Precautions
Before formal testing, it is recommended to perform a pilot experiment using 2-3 samples expected to have significant activity differences.
For tissue and cell samples, protein concentration measurement can be used to normalize results between samples.
This kit is compatible with spectrophotometer detection. Adjust the preparation volumes of detection reagents proportionally according to the spectrophotometer's requirements.
Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other protective equipment. Perform experiments in a fume hood or biosafety cabinet.
This product is for scientific research use only. It is not intended for clinical diagnosis.
Frequently Asked Questions (FAQ)
1. What should I do if the measured ΔA is too high or too low?
If ΔA > 0.5, the sample HK activity is too high. Dilute the supernatant appropriately with Extraction Buffer (include the dilution factor *n* in the calculation) or shorten the reaction time to 5 min.
If ΔA < 0.005, the sample HK activity is too low. Increase the sample volume (keep the Working Solution volume constant and adjust the variable in the formula accordingly) or extend the reaction time (e.g., to 15 or 30 min).
2. Can multiple samples be assayed simultaneously for high-throughput detection?
The initial reaction rate is fast. It is not recommended to run a large number of samples simultaneously. If a multichannel pipette is unavailable, it is best for two individuals to perform the experiment together—one person timing and the other measuring the absorbance—to ensure the accuracy of the results.
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