Phospholipase A2 (PLA2) is a calcium-dependent, heat-stable enzyme that catalyzes the hydrolysis of glycerophospholipids at the sn-2 position of cellular membranes, thereby releasing Arachidonic Acid (AA). Phospholipase A2 is a key mediator in the biosynthesis of pro-inflammatory lipid mediators, critically involved in inflammatory processes. Phospholipase A2 can be used for cardiovascular and inflammatory diseases research.
Preparation and Specification
Appearance: Colorless to brownish solution
Specific activity: More than 200 U/ml
Properties
| Substrate specificity |
| See Table 1 | | Molecular weight | 15 kDa (Sephadex G-100) |
| | Isoelectric point | pH 7.51 |
| | Michaelis constants | Lecithin 5.0 × 10⁻³ M |
| | Optimum pH | 7.3–8.3 | Figure 1 | | pH stability | 6.0–9.5 (55℃, 10 min) | Figure 2 | | Thermal stability | Stable at 50℃ and below (pH8.0, 10 min, + CaCl₂) | Figure 3 | | Stabilizer | Ca²⁺ |
| | Activators | Ca²⁺, Non-ionic detergents |
| | Inhibitor | EDTA |
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Applications for Diagnostic Test
This enzyme is useful for enzymatic determination of lecithin when coupled with lysophospholipase, glycerophosphorylcholine phosphodiesterase and choline oxidase.
Table 1. Substrate specificity
| Substrate | Relative activity (%) |
|---|
| Phosphatidylcholine | 100 | | Phosphatidylethanolamine | 30 | | Phosphatidic Acid | 10 | | Lysophosphatidylcholine | 0 | | Triolein | 0 | | Trilaurin | 0 |
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Assay
Principle
The assay is based on the increase in absorbance at 500 nm as the formation of quinoneimine dye proceeds in the following reactions:
ACS: Acyl–CoA synthetase
ACOD: Acyl–CoA oxidase
ATP: Adenosine triphosphate
Unit definition
One unit is defined as the amount of enzyme which liberates 1 μmole of fatty acid per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
1. Reaction mixture for the first reaction
0.2 M Tris-HCl buffer, pH 8.0 0.20 ml
20 mM DPPC solution ᵃ 0.05 ml
1 M CaCl₂ solution 0.025 ml
0.2 M ATP solution, pH 7.0 0.01 ml
0.1 M CoA solution, pH 6.0 ᵇ 0.02 ml
10.6 U/ml ACS solution ᶜ 0.05 ml
Distilled water 0.145 ml
a: 20 mM DPPC solutionDissolve 147 mg of DPPC with 10 ml of 2% (W/V)
Triton X–100 solution.
b: 0.1 M CoA solution pH 6.0
Dissolve 7.85 g (purity calculation) of CoA with 80 ml of distilled water and adjust pH to 6.0 (25℃) with 4 N NaOH and add distilled water to make a total of 100ml.
c: 10.6 U/ml ACS solution
Dissolve 106 U of ACS with 10 ml of Tris–HCl buffer pH 8.0.
DDPC: Dipalmytoylphosphatidylcholine
Procedure
1. Pipette accurately 0.50 ml of reaction mixture for the first reaction into a small test tube and preincubate at 37℃.
2. After 5 min, add exactly 50 μl of enzyme sample and mix to start the first reaction at 37℃.
※ In the case of a test blank, add 50 μ1 of enzyme dilution buffer in place of enzyme solution at this point.
3. After 10 min, add 0.5 ml of 20 mM NEM and add 0.5ml of reaction mixture for the second reaction after 15 seconds later and mix to start the second reaction at 37℃.
4. At 5 min after starting the reaction, add 1.5 ml of the reaction stopper to stop the reaction.
5. Measure the absorbance at 500 nm.
△A = (As−Ab) ≦ 0.20 Abs
Absorbance sample : As
blank : Ab
Calculation
Activity (U/mg) = {(△A/10)/(12.0×1/2)} × 3.05/0.05 × D
12.0 : millimolar extinction coefficient of quinoneimine dye at500 nm(cm²/µmole)
1/2 : a multiplier derived from the fact that 2 mole of H₂O₂produces 1 mole of quinoeimine dye
10 : reaction time (min)
3.05 : final volume (ml)
0.05 : volume of enzyme solution (ml)
D : times of dilution in enzyme solution
