AN AFFINITY COLUMN FOR PHOSPHOLIPASE A(2) BASED ON IMMOBILIZED ACYLAMINOPHOSPHOLIPID ANALOGS

A synthetic route was developed to prepare 2-acylamino phospholipid an alogues suitable for immobilisation. The inhibitors, synthesised in ei ther the (R)- and (S)-configuration, carried an omega-carboxyl group i n one acyl chain for immobilisation to the matrix. As a matrix Sepharo se 6B, derivatised with a polar, non-charged 16 atom spacer was used. Low-molecular weight phospholipase A(2) binds in a calcium-dependent w ay to the immobilised (S)-inhibitor and not to the immobilised (R)-inh ibitor which shows that binding involves specific active site interact ions rather than hydrophobic chromatography. The specificity was furth er demonstrated by the fact that the immobilised (S)-inhibitor binds p orcine pancreatic and snake venom phospholipases A(2), but not the por cine pancreatic zymogen. Moreover, a mutant porcine pancreatic phospho lipase A(2) in which the active side residue His48 has been replaced b y Gln, was not bound by the column. This column material might be appl icable for affinity purification of phospholipase A(2) and for screeni ng of phage display libraries. (C) 1997 Elsevier Science B.V.