Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii

Phospholipids are complex lipids containing phosphate groups. These molecules are hydrolyzed by an enzyme called phospholipase.

To perform the phospholipase activity assay, take an egg yolk emulsion rich in phospholipids, which act as substrate for the enzyme. Add the yolk to a warm agarose solution containing sodium chloride, which enhances the stability of the yolk proteins during subsequent steps.

Supplement the mixture with calcium chloride and a fluorescent dye - rhodamine. Pour the mixture into a Petri plate. Upon solidification, the yolk phospholipids remain homogeneously distributed in the agarose gel matrix.

Using a tube, create appropriately-distanced wells in the gel. Add varying concentrations of crude protein extract containing phospholipase to the wells. Incubate the plate at physiological temperature.

The phospholipase from the extract diffuses radially into the gel. Calcium ions in the gel bind to the phospholipase, activating the enzymes. This enzyme cleaves the phospholipids, generating free fatty acids. The rhodamine molecules in the gel form a complex with the released fatty acids.

Upon illumination with ultraviolet light, observe the appearance of fluorescent halos around the wells. The halo confirms the presence of enzyme activity. With higher concentrations of phospholipase, the enzyme molecules diffuse further, increasing the diameter of the halo.

Wash one chicken egg with 1% SDS in distilled water, and separate the egg yolk from the egg white under sterile conditions.

Prepare solutions A, B, C, and D. Add 500 microliters of solutions A and C to solution B, and 100 microliters of solution D. Mix them and pour 25 milliliters into each Petri dish. Wait for the solution to solidify under sterile conditions, and make wells of approximately 2 to 3 millimeters in diameter, with a thin tube.

Add a total of 20 microliters of phosphate buffer as a negative control in one well, and 20 microliters of a determined phospholipase as a positive control in another well.

Place different amounts of the crude extract protein - 5, 15, 25, 35, and 45 micrograms in the remaining wells, each in a final volume of 20 microliters. Incubate at 37 degrees Celsius for 20 hours.