MOBILE-PHASE EFFECTS ON MEMBRANE-PROTEIN ELUTION DURING IMMOBILIZED ARTIFICIAL MEMBRANE CHROMATOGRAPHY

The eluotropic strength of different mobile phases for eluting membran e proteins from immobilized artificial membrane (IAM) chromatography s urfaces was studied. Two protein mixtures containing bovine pancreatic PLA(2) were used in this study. Protein mixture I was PLA(2) obtained from Sigma which contained similar to 5-10 major protein bands in ele ctrophoretic gels. Protein mixture II was obtained from fresh bovine p ancreatic tissue and contained >100 proteins including the target prot ein, PLA(2). After adsorbing Sigma PLA(2) to IAM columns, the elution conditions common to conventional chromatographic methods were evaluat ed for their ability to selectively purify PLA(2). Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during i on-exchange chromatography, (iii) salt conditions used during hydropho bic interaction chromatography, (iv) acetonitrile gradients used durin g reversed-phase chromatography, and (v) a two-step gradient consistin g of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels, PLA(2) from prot ein mixture I was purified to electrophoretic homogeneity with 417-fol d increase in specific activity in one step using elution condition (v ), and PLA(2) from protein mixture II was purified in one step (660-fo ld increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%.