Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2 (original) (raw)

A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [ 1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A,, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission a t 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A,, the emission maximum shifted to about 470 nm. P P C showed a critical micellar concentration around 5 pM, with evidence of premicellar aggregation above 1 pM. Binding of P P C to Crotalus adamanteus phospholipase A, was evidenced by a n increase in emission a t 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 pM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants

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