BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
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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