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Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.
Assay Principle
ACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.
| Component | 50T | Storage |
| Extraction Buffer | 80 mL | 2-8℃ |
| Reagent 1 | 1EA | -20℃ |
| Reagent 2 | 4EA | -20℃ |
| Reagent 3 | 2EA | -20℃ |
| Reagent 4 | 2EA | -20℃ |
Reagent Preparation
Reagent 1: Before use, centrifuge the vial to bring the powder to the bottom. Add 8.4 mL of distilled water to dissolve. Aliquot and store unused portions at -20°C.
Reagent 2: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.
Reagent 3: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.
Reagent 4: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.
Required Materials and Equipment (Not Provided)
UV spectrophotometer, 1 mL quartz cuvette (1 cm light path), refrigerated benchtop centrifuge, constant temperature incubator, pipettes, mortar and pestle, ice, and distilled water.
Sample Preparation
1.Tissue Samples: Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 12,000 rpm (approx. 13,000-15,000 g), 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Note: For larger samples, use an Extraction Buffer volume (mL) to tissue mass (g) ratio between 5:1 and 10:1.
2.Bacteria/Cell Samples: Collect cells by centrifugation and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million cells/bacteria. Disrupt the cells/bacteria by sonication on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 12,000 rpm, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.
Note: For larger samples, use an Extraction Buffer volume (mL) to cell count (10⁴ cells) ratio between 1:1000 and 1:5000.
Assay Procedure:
1.Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.
2.Pre-warm all reagents at 37°C for 5-15 minutes.
3.Optional Master Mix: A Master Mix can be prepared just before use by combining Extraction Buffer, Reagent 1, Reagent 2, Reagent 3, and Reagent 4 in a 400:160:40:20:20 ratio (e.g., for one assay: 400μL + 160μL + 40μL + 20μL + 20μL = 640μL). Pipette 640 μL of this Master Mix per cuvette. Prepare the Master Mix fresh for immediate use.
4.Pipette into a 1 mL quartz cuvette (1 cm light path) in the following order:
| Reagent | Volume (μL) |
| Extraction Buffer | 400 |
| Reagent 1 | 160 |
| Reagent 2 | 40 |
| Reagent 3 | 20 |
| Reagent 4 | 20 |
| Mix thoroughly and incubate at 37°C for 5 min. | |
| Sample | 60 |
Mix thoroughly immediately after adding the sample. Record the initial absorbance (A₁) at 340 nm at 10 seconds. Record the absorbance again (A₂) after exactly 10 minutes. Calculate ΔA = A₁ - A₂.
Notes & Troubleshooting:
1.If ΔA is close to zero, consider extending the reaction time (e.g., to 20 min) to read A₂. Use the new reaction time (T) in the calculation. Alternatively, increase the sample volume V₁ (e.g., to 100 μL, decrease Extraction Buffer accordingly) and use the new V₁ and T in the calculation.
2.If the initial absorbance A₁ is too high (e.g., >2, common in deeply pigmented plant samples), reduce the sample volume V₁ and use the new V₁ in the calculation. Alternatively, add a small amount of activated charcoal to the sample supernatant, mix, let stand for 5 min, centrifuge at 12,000 rpm, 4°C for 10 min, and use the clarified supernatant for assay.
3.If ΔA is greater than 0.6, reduce the reaction time (e.g., to 5 min) and use the new time (T) in the calculation.
4.If the decrease is not linear, read the absorbance every 10 seconds and select a linear segment for calculating ΔA. Use the corresponding time interval (T) for the calculation.
ACK Activity Calculation:
General Parameters:
ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cm
d (Cuvette light path) = 1 cm
V (Total extraction volume) = 1 mL
V₁ (Sample volume in reaction) = 0.06 mL (60 μL)
V₂ (Total reaction volume) = 0.0007 L (700 μL)
T (Reaction time) = 10 min
500 (Cell/Bacteria count in ten-thousands: 500 × 10⁴ = 5 million)
W (Sample mass, g)
Cpr (Sample protein concentration, mg/mL)
1. 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:
ACK Activity (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (Cpr × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/mg prot) = 187.6 × ΔA ÷ Cpr
2. 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 fresh tissue.
Calculation:
ACK Activity (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/g fresh weight) = 187.6 × ΔA ÷ W
3. Based on Bacterial/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/bacteria.
Calculation (for 5 million cells in 1 ml extract):
ACK Activity (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ / V) ÷ T
Simplified Formula: ACK (nmol/min/10⁴ cell) = 0.38 × ΔA
Precautions
Before formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.
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