Abstract
A rapid and continuous method for measuring phospholipase A 2 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 A 2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A 2 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 μl. This new method should be useful for assaying phospholipase A 2 activities in various biological systems.
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