Phospholipase A2 is a member of the class of heat-stable, calcium-dependent enzymes catalyzing the hydrolysis of the 2-acyl bond of 3-n-phosphoglycerides. The enzyme has a molecular weight of 30,000 daltons. Phospholipase A2 is activated by Ca2+. It is inhibited by zinc, barium, and manganese ions. Activity values for phospholipase A preparations which are derived from titrimetric assay procedures can be quite dependent on source and type of lecithin, its preparation as a substrate emulsion, other components of the reaction mixture, and the method and instrumentation used.
Phospholipase A2 represents a class of heat-stable, calcium-dependent enzymes catalyzing the hydrolysis of the 2-acyl bond of 3-n-phosphoglycerides. This enzyme is named Phospholipase A2 to denote its 2-acyl specificity.
Phospholipase A2 has been isolated from pancreas, snake and bee venoms.
Phospholipases are involved in lipid metabolism and are important probes of structure-function relationships in biological membranes.
Specificity
The enzyme specifically catalyzes the hydrolysis of the β-fatty ester linkage of L-α phosphoglycerides.
Composition
Two proteins (A2-α, A2-β) exhibiting Phospholipase A activity have been isolated from Crotalus adamanteus. Each protein is composed of dimeric subunits. The two proteins have similar activities but are chromatographically and electrophoretically distinct. Heinrikson et al. (1977) report on the distinctive structural features of the two enzymes and the complete amino acid sequence of phospholipase A2-α. The two subunits differ at a single amino acid; A2-α contains glutamine at residue 117, while the more acidic A2-β contains glutamic acid at this residue.
Characteristics of Phospholipase A2
Protein Accession Number: P00623
Molecular weight: 30,000
Extinction coefficient: extinction coefficient = 22.7
Isoelectric point: 4.55 and 4.40 for A-α and A-β, respectively
Activators: Ca²⁺
Inhibitors: Zn²⁺, Ba²⁺, Mn²⁺
Phospholipase A2 Assay
Method
Activity values for phospholipase A2 preparations which are derived from titrimetric assay procedures can be quite dependent on source and type of lecithin, its preparation as a substrate emulsion, other components of the reaction mixture, and the methodology/instrumentation utilized. Values reported in the literature for highly purified venom phospholipase A2 have ranged from 200 to 3000 units/mg. Comparison between assay systems is difficult. The following assay has been found to be reproducible in our laboratory. One unit releases one micromole titratable fatty acid per minute from lecithin emulsion at pH 8.9 and 25°C under the specified conditions.
Reagents
0.1 M Calcium chloride
1.0 M Sodium chloride
Lecithin emulsion: Weigh out 4.0 grams reagent grade soybean lecithin in a 250 ml beaker and add 30 ml of 1.0 M sodium chloride, 10 ml of 0.1 M calcium chloride and 100 ml of reagent grade water. After stirring for 30 minutes at 4°C, sonicate the mixture for 10 minutes at maximum power on a Branson sonicator or its equivalent. Dilute with reagent grade water to a final volume of 200 ml.
0.01-0.02 N NaOH - Standardized
Enzyme
Dissolve enzyme at a concentration of 1.0 mg/ml in reagent grade water. Keep this stock solution on ice while assay is being run. Further dilutions are made in reagent grade water immediately prior to use.
Procedure
The titration can he carried out with an automatic titrator or on a laboratory pH meter. The reaction vessel should be maintained at 25°C.
Blank rate determination: Pipette 15 ml of lecithin emulsion into a reaction vessel at 25°C. Adjust the pH to 8.9 and record the volume of titrant required to maintain the pH at 8.9 for 3-5 minutes after a constant rate is obtained. Determine the "blank rate" as the volume of titrant added per minute from the final linear portion of the curve.
Sample determination: Add appropriately diluted enzyme to the above emulsion. Record the amount of titrant required to maintain the pH at 8.9 for 4-5 minutes. Determine the "sample rate" as the volume of titrant added per minute from the linear portion of the curve.
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