Description
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 PrinciplePK 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.Component50TStorageExtraction Buffer60 mL2-8℃Reagent A50 mL2-8℃Reagent B 2EA-20℃Reagent C25 µL×22-8℃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 Preparation1.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 Procedure1.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 sample50 µl WR III900 µl WR IIMix 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 CalculationGeneral Formula:PK Activity = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ ÷ Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TWhere:Vₜₒₜₐₗ = Total reaction volume = 0.000975 L (975 µl)ε = NADH molar extinction coefficient = 6220 L/mol/cmd = Light path length = 1 cm10⁹ = Conversion factor from moles to nanomoles (nmol)T = Reaction time = 2 minVₛₐₘₚₗₑ = Sample volume added to reaction = 0.050 mlVₛₐₘₚₗₑₜₒₜₐₗ = Total volume of extract used = 1 ml (for tissues/cells)Cpr = Sample protein concentration (mg/ml)W = Sample mass (g)500 = Cell/Bacteria count (in millions, for the example calculation: 5 million = 500 × 10⁴)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 ÷ 2Simplified Formula: PK (nmol/min/ml) = 2613 × ΔA2. 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) ÷ 2Simplified Formula: PK (nmol/min/mg prot) = 2613 × ΔA ÷ Cprb. 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) ÷ 2Simplified Formula: PK (nmol/min/g fresh weight) = 2613 × ΔA ÷ Wc. 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) ÷ 2Simplified Formula: PK (nmol/min/10⁴ cell) = 5.226 × ΔA