A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.
1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.
Wilton
D C
DC
Department of Biochemistry, School of Biochemical and Physiological Sciences, University of Southampton, U.K.
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D066298:In Vitro Techniques; D008049:Lipase / Q000032:analysis* / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence; D013552:Swine
D002352:Carrier Proteins; D003619:Dansyl Compounds; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C054400:11-(dansylamino)undecanoic acid; D010740:Phospholipases; D008049:Lipase; D010741:Phospholipases A; D054467:Phospholipases A2