Acyl-CoA synthetase catalyzes the activation reaction of fatty acids. In the presence of ATP, CoA and Mg²⁺, it can catalyze the activation of fatty acids to form acyl-CoA.
Application: Suitable for the enzymatic determination of free fatty acid content. Each laboratory shall determine the optimal working titer for its specific application. Other applications have not been tested, but their use in such assays should not be excluded.
Assay Procedure
Principle
Unit Definition
One unit of enzyme activity unit is equal to the amount of enzyme required to convert 1 micromole of palmitic acid in 1 minute under the following test conditions.
Reagents
Reagent A: Prepare a 0.05M potassium phosphate buffer solution with pH 7.5.
Reagent B: Add 0.6053 g of ATP·Na₂, 0.2033 g of hexahydrate magnesium chloride, and 0.8216 g of Coenzyme A to 0.8 L of Reagent A, adjust pH to 7.5, and adjust to 1 L with Reagent A. (Preparation is optional; Coenzyme A is unstable and can be added as needed.)
Reagent C: Add 50 g Triton X-100 and 2 mmol palmitic acid to 0.8 L of water. Heat to dissolve the palmitic acid completely, then cool to room temperature. Adjust pH to 7.5 with sodium hydroxide and adjust to 1 L with Reagent A (preparation by proportion).
Reagent D: Add 0.1251 g NEM (maleimide), 0.505 g 4-aminotetraphenylpyrimidine, 8kU peroxidase, and 0.25 g sodium azide to 0.8 L of Reagent A, adjust pH to 7.5, and adjust to 1 L with Reagent A (preparation by proportion).
Reagent E: Add 2 grams of phenol to 80ml of Reagent A and adjust the volume to 100ml with Reagent A (preparation by proportion).
Reagent F: ACS Dilution Buffer: Add 2 mM ATP and 1 g Triton X-100 to 1 L of 10 mM potassium phosphate buffer at pH 7.5. (Preparation can be scaled up proportionally).
Reagent G: ACOD Dilution Buffer: Add 0.02 mmol FAD and 0.2 g bovine serum albumin to 1 L of 25 mM potassium phosphate buffer at pH 7.5. (Preparation by proportion is possible).
Reagent H: Prepare a 240 kU/L ACOD (acetyl-CoA oxidase) enzyme solution using Reagent G.
Test sample: Weigh the required enzyme in a Weighing tube before testing. When weighing the enzyme, at least 10 mg of enzyme should be weighed to ensure accuracy. Dissolve the enzyme in diluent (Reagent F) and adjust to the specified volume.
Procedure
1. Mix 320μl of Reagent B with 80μl of Reagent C and incubate at 37℃ for 1 minute.
2. Add 20μl of diluted ACS enzyme solution and incubate at 37℃ for 10 minutes.
3. Add 800μl of Reagent D and 20μl of Reagent E, incubate at 37℃ for 2 minutes; then add 20μl of Reagent H and incubate at 37℃ for 5 minutes, recording the absorbance value (As).
4. Repeat steps 1-3, but replace the ACS enzyme solution with 20 μL of ACS dilution. Record the blank absorbance value (Ab).
Note: △A=As-Ab≤0.300; the optimal linear range for this method is 100-200 U/L.
Calculation
The enzyme activity is calculated using the following formula:
Vt: Total volume of the reaction mixture (1.26ml)
ε: The generated quinuimine exhibits an extinction coefficient at 500 nm of 12 (cm²/μmol)
Vs: Volume of the enzyme solution to be tested (ml)
d: Light path length of the cuvette (cm)
D: Dilution factor of the enzyme solution
△A/min: As-Ab
ZJ26F0434113