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。
丙酮酸激酶（PK）广泛存在于动物、植物、微生物和培养细胞中，催化糖酵解过程中的最后一步反应，是糖酵解过程中的主要限速酶之一，也是产生ATP的关键酶之一，因此测定PK活性具有重要意义。
测定原理： PK催化磷酸烯醇式丙酮酸和ADP生成ATP和丙酮酸，乳酸脱氢酶进一步催化NADH和丙酮酸生成乳酸和NAD+，在340nm下测定NADH下降速率，即可反映PK活性。
| Component | 50T | Storage |
| 提取液 | 60 mL | 2-8℃ |
| 试剂一 | 50 mL | 2-8℃ |
| 试剂二 | 2EA | -20℃ |
| 试剂三 | 25 μL×2 | 2-8℃ |
自备仪器和用品
紫外分光光度计、台式离心机、水浴锅、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）；8000g 4℃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。8000g4℃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、样本测定
（1）试剂二的配制：临用前取试剂二一瓶，加入22.5ml试剂一和2.65ml蒸馏水充分溶解，置于37℃（哺 乳动物）或25℃（其它物种）水浴5min；现配现用。
（2）试剂三的配制：临用前取试剂三一支，加入1.5ml蒸馏水充分溶解待用；现配现用。
（3）在1ml石英比色皿中加入50μl样本、50μl试剂三和900μl试剂二，混匀，立即记录340nm处20s时 的吸光值A1和 2min20s后的吸光值A2，计算ΔA=A1-A2。
PK活性计算:
1、血清（浆）中PK活力的计算：
单位的定义：每毫升血清（浆）每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK（nmol/min/ml）＝[ΔA×V 反总÷（ε×d）×10⁹]÷V样÷T=2613×ΔA
2、组织、细菌或细胞中PK活力的计算:
（1）按样本蛋白浓度计算：
单位的定义：每mg组织蛋白每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK（nmol/min /mg prot）＝[ΔA×V 反总÷（ε×d）×10⁹]÷(V 样×Cpr) ÷T=2613×ΔA÷Cpr
（2）按样本鲜重计算：
单位的定义：每g组织每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK（nmol/min /g 鲜重）＝[ΔA×V反总÷（ε×d）×10⁹]÷(W×V样÷V样总)÷T=2613×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消耗1nmol的NADH定义为一个酶活力单位。
PK（nmol/min /10⁴ cell）＝[ΔA×V 反总÷（ε×d）×109]÷(500×V 样÷V 样总)÷T=5.226×ΔA
V反总：反应体系总体积，9.75×10⁻⁴L；
ε：NADH摩尔消光系数，6.22×10³L/mol/cm；
d：比色皿光径， 1cm；
V样：加入样本体积，0.03ml；
V样总：加入提取液体积，1ml；
T：反应时间，2min；
Cpr：样本蛋白质浓度，mg/ml；
W：样本质量，g；
500：细菌或细胞总数，500万。
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。
Pyruvate Kinase (PK) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis, serving as one of the key rate-limiting enzymes in this process and a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.
Assay Principle
PK catalyzes the conversion of Phosphoenolpyruvate (PEP) and ADP to ATP and Pyruvate. Lactate Dehydrogenase (LDH) further catalyzes the reaction between NADH and Pyruvate to produce Lactate and NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PK activity.
| Component | 50T | Storage |
| Extraction Buffer | 60 mL | 2-8℃ |
| Reagent A | 50 mL | 2-8℃ |
| Reagent B | 2EA | -20℃ |
| Reagent C | 25 μL×2 | 2-8℃ |
Required Materials and Equipment (Not Provided)
UV spectrophotometer, benchtop centrifuge, water bath, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.
Sample Preparation
1.Bacteria or Cultured Cells:
Collect bacteria or cells into a centrifuge tube, centrifuge, 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/bacteria).
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.
2.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.
3.Serum (or Plasma) Samples:
Assay directly.
Assay Procedure
1.Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.
2.Sample Assay:
(1) Preparation of Working Reagent II (WR II): Just before use, dissolve the contents of one vial of Reagent B in 22.5 ml of Reagent A and 2.65 ml distilled water. Mix thoroughly. Incubate WR II at 37°C (for mammalian samples) or 25°C (for other species) in a water bath for 5 minutes. Prepare fresh for each use.
(2) Preparation of Working Reagent III (WR III): Just before use, dissolve the contents of one tube of Reagent C in 1.5 ml distilled water. Mix thoroughly. Prepare fresh for each use.
(3) Reaction Setup: In a 1 ml quartz cuvette, add:
50 μl sample
50 μl WR III
900 μl WR II
Mix thoroughly and immediately record the absorbance (A₁) at 340 nm at 20 seconds. Record the absorbance again (A₂) after 2 minutes and 20 seconds (140 seconds total). Calculate ΔA = A₁ - A₂.
PK Activity Calculation
General Formula:
PK Activity = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ ÷ Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T
Where:
1. For Serum (Plasma):
Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per ml of serum.
Calculation:
PK Activity (nmol/min/ml) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ 0.050 ÷ 2
Simplified Formula: PK (nmol/min/ml) = 2613 × 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 consumes 1 nmol of NADH per minute per mg of protein.
* Calculation:
PK Activity (nmol/min/mg prot) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (0.050 × Cpr) ÷ 2
Simplified Formula: PK (nmol/min/mg prot) = 2613 × ΔA ÷ Cpr
b. 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 tissue.
* Calculation:
PK Activity (nmol/min/g fresh weight) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (W × 0.050 / 1) ÷ 2
Simplified Formula: PK (nmol/min/g fresh weight) = 2613 × ΔA ÷ W
c. Based on Bacterial or 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.
* Calculation (using the example of 5 million cells extracted in 1 ml):
PK Activity (nmol/min/10⁴ cell) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (5 × 0.050 / 1) ÷ 2
Simplified Formula: PK (nmol/min/10⁴ cell) = 5.226 × ΔA
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