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。
己糖激酶（HK）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，催化葡萄糖转化为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。
测定原理： 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 | 50T | Storage |
| 提取液 | 60 mL | 2-8℃ |
| 试剂一 | 30 mL | 2-8℃ |
| 试剂一 | 1EA | 2-8℃ |
| 试剂一 | 5 mL | 2-8℃ |
| 试剂一 | 1EA | -20℃ |
| 试剂一 | 1EA | -20℃ |
| 试剂一 | 1EA | -20℃ |
试剂二：粉剂×1瓶，4℃保存；临用前加入30ml蒸馏水充分溶解备用；用不完的试剂分装后-20℃保存，禁止反复冻融。
试剂四：粉剂×1支，-20℃保存，临用前加入4ml蒸馏水充分溶解备用；用不完的试剂分装后-20℃保存，禁止反复冻融。
试剂五：粉剂×1支，-20℃保存；临用前加入2ml蒸馏水充分溶解备用；用不完的试剂分装后-20℃保存，禁止反复冻融。
试剂六：粉剂×1支，-20℃保存；临用前加入250μl试剂一和250μl蒸馏水充分溶解备用；用不完的试剂分装后-20℃保存，禁止反复冻融。
自备仪器和用品
紫外分光光度计、恒温水浴锅、台式离心机、可调式移液器、1ml石英比色皿、研钵、冰和蒸馏水。
样本的前处理：
1、细菌或培养细胞：先收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量（104个）：提取液体积（ml）为500~1000：1的比例（建议500万细菌或细胞加入1ml提取液），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）；8000g4℃离心10min，取上清，置冰上 待测。
2、组织：按照组织质量（g）：提取液体积(ml)为1：5~10的比例（建议称取约0.1g组织，加入1ml提取 液），进行冰浴匀浆。8000g 4℃离心10min，取上清，置冰上待测。
3、血清（浆）样品：直接检测。
测定步骤：
1、分光光度计预热30min以上，调节波长至340nm，蒸馏水调零。
2、将试剂一、二、三、四和五37℃(哺乳动物)或25℃(其它物种)预热10分钟。
3、加样表
| 试剂（μL） | 测定管 |
| 试剂一 | 400 |
| 试剂二 | 400 |
| 试剂三 | 80 |
| 试剂四 | 80 |
| 试剂五 | 40 |
| 试剂六 | 8 |
| 样本 | 30 |
将上述试剂按顺序加入1ml石英比色皿中，立即混匀，加样本的同时开始计时，在340nm波长下记录20秒时的初始吸光度A1和5分20秒时的吸光度A2，计算ΔA=A2-A1。
注意事项：
1、为了减小操作误差，建议将试剂一、二、三、四、五按比例配成混合液，预热10min，取30μl样本+8μl 试剂六+1ml混合液，混匀后按照上述步骤检测。
2、不同匀浆组织中HK活力不一样，做正式试验之前请做1-2只预试，若ΔA>0.5，则说明组织活力太高， 必须用提取液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2min,使 ΔA<0.5，以提高检测灵敏度。
HK活性计算：
1、血清（浆）HK活性
单位的定义：每毫升血清（浆）在每分钟生成1nmol的NADPH定义为一个酶活力单位。
HK（nmol/min/ml）＝[ΔA×V 反总÷（ε×d）×10⁹]÷V样÷T=1113×ΔA
2、组织、细菌或细胞中HK活性
（1）按样本蛋白浓度计算
单位的定义：每mg组织蛋白每分钟生成1nmol的NADPH定义为一个酶活力单位。
HK（nmol/min /mg prot）＝[ΔA×V 反总÷（ε×d）×10⁹]÷(V样×Cpr)÷T=1113×ΔA÷Cpr
（2）按样本鲜重计算
单位的定义：每g组织每分钟生成1nmol的NADPH定义为一个酶活力单位。
HK（nmol/min /g 鲜重）＝[ΔA×V反总÷（ε×d）×10⁹]÷(W×V样÷V样总)÷T=1113×ΔA÷W
（3）按细菌或细胞密度计算
单位的定义：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。
HK（nmol/min /10
⁴
cell）＝[ΔA×V 反总÷（ε×d）×10⁹]÷(500×V样÷V样总)÷T=2.226×ΔA
参数说明：
V反总：反应体系总体积=1.038×10⁻³L
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。
Introduction
Hexokinase (HK) 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 is 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 | 50T | Storage |
| Extraction Buffer | 60 mL | 2-8℃ |
| Reagent 1 | 30 mL | 2-8℃ |
| Reagent 2 | 1EA | 2-8℃ |
| Reagent 3 | 5 mL | 2-8℃ |
| Reagent 4 | 1EA | -20℃ |
| Reagent 5 | 1EA | -20℃ |
| Reagent 6 | 1EA | -20℃ |
Reagent 2: Powder × 1 bottle. Dissolve in 30 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Reagent 4: Powder × 1 tube. Dissolve in 4 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Reagent 5: Powder × 1 tube. Dissolve in 2 mL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Reagent 6: Powder × 1 tube. Dissolve in 250 μL Reagent 1 and 250 μL distilled water before use. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.
Required Materials and Equipment (Not Provided)
UV spectrophotometer, constant temperature water bath, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.
Sample Preparation:
Bacteria or Cultured Cells:
Collect cells by centrifugation and discard the supernatant.
Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).
Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).
Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Tissues:
Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).
Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Serum (or Plasma) Samples:
Assay directly.
Assay Procedure:
Preheat the spectrophotometer for at least 30 min. Set wavelength to 340 nm. Zero with distilled water.
Pre-warm Reagents 1, 2, 3, 4, and 5 to 37°C (for mammalian samples) or 25°C (for other species) for 10 min.
Pipette into a 1 ml quartz cuvette in the following order:
| Reagent | Volume (μL) |
| Reagent 1 | 400 |
| Reagent 2 | 400 |
| Reagent 3 | 80 |
| Reagent 4 | 80 |
| Reagent 5 | 40 |
| Reagent 6 | 8 |
| Sample | 30 |
Mix immediately upon sample addition and start the timer.
Record the initial absorbance (A₁) at 20 seconds and the final absorbance (A₂) at 5 minutes and 20 seconds (320 sec total) at 340 nm.
Calculate ΔA = A₂ - A₁.
Notes:
To minimize operational error, it is recommended to pre-mix Reagents 1, 2, 3, 4, and 5 in the stated proportions. Pre-warm this Master Mix for 10 min. Then add 30 μl sample + 8 μl Reagent 6 + 1 ml Master Mix to the cuvette. Mix and proceed with the assay.
HK activity varies across different tissues. Perform a pilot test with 1-2 samples before formal assay. If ΔA > 0.5, the tissue activity is too high. Dilute the supernatant with Extraction Buffer (include dilution factor D in calculations) or shorten the reaction time to 2 min to ensure ΔA < 0.5 and improve detection sensitivity.
HK Activity Calculation:
General Parameters:
Vₜₒₜₐₗ (Total reaction volume) = 1.038 × 10⁻³ L (1038 μL)
ε (NADPH molar extinction coefficient) = 6.22 × 10³ L/mol/cm
d (Cuvette light path) = 1.0 cm
Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.03 mL (30 μL)
T (Reaction time) = 5 min
Cpr (Sample protein concentration, mg/mL)
W (Sample mass, g)
Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1.0 mL (for tissue/cell calculations)
500 (Cell/Bacteria count in millions for example calculation: 5 million)
1. For Serum (Plasma):
Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per ml of serum.
Calculation:
HK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ T
Simplified Formula: HK (nmol/min/ml) = 1113 × ΔA
2. For Tissues, Bacteria, or Cells:
a. Based on Sample Protein Concentration:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per mg of protein.
* Calculation:
HK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T
Simplified Formula: HK (nmol/min/mg prot) = 1113 × ΔA ÷ Cpr
b. Based on Sample Fresh Weight:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of fresh tissue.
* Calculation:
HK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: HK (nmol/min/g fresh weight) = 1113 × ΔA ÷ W
c. Based on Bacterial or Cell Density:
* Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells.
* Calculation (example for 5 million cells in 1 ml extract):
HK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Simplified Formula: HK (nmol/min/10⁴ cell) = 2.226 × ΔA
Precautions
Perform a pilot assay with 2-3 samples expected to have significant activity differences before the formal determination.
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