Affinity purification of phospholipase A(2) on immobilized artificial membranes containing and lacking the glycerol backbone

Immobilized artificial membranes (IAMs) are chromatography surfaces containing monolayers of phospholipid ligands. (ether)IAM.PCC10/C3 contains the glycerol backbone whereas (delta G)IAM.PCC10/C3 lacks the glycerol backbone. Affinity purification of PLA(2) on these IAM surfaces demonstrated that the surface structural differences were not important for phospholipase A(2) (PLA(2)) binding. This suggests that the chromatographically important binding event involves the PLA(2) surface and the monolayer of polar choline headgroups on the IAM surface. After sample loading, short-chain alkylsulfonates were used as low eluotropic strength detergents to remove contaminating proteins, and PLA(2) were eluted with CH3CN (30%). Octyllysophosphatidylcholine (0.5%) can replace CH3CN to elute PLA(2) from IAM surfaces, The PLA(2) purity after IAM chromatography depends on the protein loading; analytical-scale loadings (0.8 mg protein/g IAM) resulted in a PLA(2) purity of ca. 70% based on densitometric scans of proteins in polyacrylamide gels after electrophoresis. Preparative loadings of 3.21 mg protein/g IAM resulted in 48% PLA(2) purity. Purification of PLA(2) to electrophoretic homogeneity was achieved using an IAM column followed by a strong anion-exchange column. These results suggests that IAMs may be used to develop purification methods for PLA(2) enzymes obtained from diverse biological specimens.