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