1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine, a highly specific chromogenic substrate of phosphol ipase A 2

Abstract

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine (2-thioPC), a structurally modified phospholipid analog is specifically hydrolyzed by phospholipase A 2 to liberate 2-thiolysophosphatidylcholine and palmitic acid. The sulfhydryl group of the product is readily trapped by 5,5′-dithiobis (2-nitrobenzoic acid) allowing continuous spectrophotometric monitoring of the enzymatic reaction. The rates of hydrolysis by bee-venom phospholipse A 2 have been determined in a series of Triton X-100 containing mixed micelles. At 1 mM 2-thioPC increasing the concentration of Triton X-100 from 4 to 16 mM changes the specific activity of bee-venom phospholipase A 2 from 96.9 to 17.9 μmol/min/mg, about one order of magnitude lower than dipalmitoyl phosphatidylcholine hydrolysis in micelles of similar composition. The chromogenic substrate is the first phospholipid analog exhibiting absolute specificity for phospholipase A 2 and should be applicable to spectrophotometric detection and kinetic characterization of both water soluble and membrane-bound forms.