Spin-label assay for phospholipase A2.
A rapid and continuous method for measuring phospholipase A2 activity using electron spin resonance spectroscopy and a spin-labeled phospholipid as a substrate has been developed. The substrate, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine, gives rise principally to a broad ESR line in aqueous solution due to strong spin-spin interactions, probably resulting from its micellar formation. Upon addition of bee venom phospholipase A2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A2 activity can be followed by monitoring the increase in the ESR signal amplitude of the three-line spectrum, which is linearly proportional to the amount of 4-doxylpentanoic acid produced; no separation of the product from the substrate is needed during the measurement. The rate of hydrolysis of 1 nmol min-1 can be accurately measured within a 5-min period of time in a sample volume of 100 microliters. This new method should be useful for assaying phospholipase A2 activities in various biological systems.
Lai
C S
CS
Department of Radiology, Medical College of Wisconsin, Milwaukee 53226.
Zhang
J Z
JZ
Joseph
J
J
D001514:Bee Venoms / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D008246:Lysophospholipids / Q000032:analysis; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D010743:Phospholipids / Q000032:analysis
D001514:Bee Venoms; D008246:Lysophospholipids; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2