A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin.
Phospholipase A2 activity can be determined fluorometrically in the presence of serum albumin using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid. In the water reaction medium 10-pyrene phospholipids form vesicles and the monomer fluorescence of the pyrene is negligible due to pyrene-pyrene interaction. Upon phospholipid hydrolysis 10-pyrenyldecanoic acids are produced and tightly bind to albumin so that a monomer pyrene fluorescence is observed. We obtained an excellent parallelism between hydrolysis determined by a classical extraction method and that followed by direct and continuous spectrofluorometric recording of the monomer emission of pyrene. This assay can measure picomole amounts of phospholipids hydrolyzed per minute so that picogram quantities of phospholipases A2 from pancreas or from venoms can be measured. Phospholipase activity remains proportional to enzyme concentration over three orders of magnitude. The method can be used to quantify the phospholipase A2 activity of crude extracts of low specific activity.
Radvanyi
F
F
Laboratoire des Venins, Unité associée Institut Pasteur/INSERM No. 285, Paris, France.
Jordan
L
L
Russo-Marie
F
F
Bon
C
C
D006868:Hydrolysis; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D010743:Phospholipids; D011721:Pyrenes; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence
D010743:Phospholipids; D011721:Pyrenes; D012710:Serum Albumin, Bovine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2