Affinity purification of phospholipase A2 on immobilized artificial membranes containing and lacking the glycerol backbone.
Immobilized artificial membranes (IAMs) are chromatography surfaces containing monolayers of phospholipid ligands. etherIAM.PCC10/C3 contains the glycerol backbone whereas delta GIAM.PCC10/C3 lacks the glycerol backbone. Affinity purification of PLA2 on these IAM surfaces demonstrated that the surface structural differences were not important for phospholipase A2 (PLA2) binding. This suggests that the chromatographically important binding event involves the PLA2 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 PLA2 were eluted with CH3CN (30%). Octyllysophosphatidylcholine (0.5%) can replace CH3CN to elute PLA2 from IAM surfaces. The PLA2 purity after IAM chromatography depends on the protein loading; analytical-scale loadings (0.8 mg protein/g IAM) resulted in a PLA2 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% PLA2 purity. Purification of PLA2 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 PLA2 enzymes obtained from diverse biological specimens.
Bernal
C
C
Department of Medicinal Chemistry, School of Pharmacy, Perdue University, West Lafayette, IN 47907, USA.
Pidgeon
C
C
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000737:chemistry*; D008567:Membranes, Artificial; D010741:Phospholipases A / Q000302:isolation & purification*; D054467:Phospholipases A2
D003435:Crotalid Venoms; D008567:Membranes, Artificial; D010741:Phospholipases A; D054467:Phospholipases A2; D005990:Glycerol