Carbonothioate phospholipids as substrate for a spectrophotometric assay of phospholipase A(2)

A continuous spectrophotometric assay for phospholipase A(2) (PLA(2)) was d eveloped using novel carbonothioate phospholipids. These phospholipid analo gues contain a carbonothioate bond in the place of the sn-2 ester of the na tural substrates of phospholipase A(2) and were synthesized in a one-pot tw o-step reaction. Phospholipase A(2) from cobra venom (Naja naja atra) hydro lyzes carbonothioate phospholipids and liberates a free thiol, alkylmercapt an, which is reacted with 5,5'-dithiobis(2-nitrobenzoic acid) to yield a pr oduct that absorbs at 412 mo. The kinetic studies on PLA(2) hydrolysis of c arbonothioate phospholipids were carried out in pure phospholipid forms and in Triton X-100 mixed micelles. The hydrolysis of pure carbonothioate phos pholipids exhibits an interfacial activation phenomenon. The hydrolysis of phospholipid in mixed Triton X-100 micelles follows classical Michaelis-Men ten kinetics. In a mixed micellar system, the catalytic efficiency observed with this series of substrates is two orders of magnitude lower than that of the hydrolysis of the natural substrate dipalmitoyl phosphocholine. Howe ver, these substrates bind to the enzyme over 10 times tighter than does th e natural substrate. Application of this carbonothioate assay to screen bot h reversible and irreversible enzyme inhibitors of phospholipase A(2) is al so demonstrated. (C) 1998 Academic Press.