Lateral interaction between electrostatically adsorbed and covalently immobilized proteins on the surface of cation-exchange sorbents.
This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized proteins coats 50% or less of the sorbent surface. It was found that although these sorbents still function as cation exchangers, covalently immobilized proteins frequently contribute to the ion-exchange behavior of some protein analytes. Chromatographic retention of analytes was equal to or greater on immobilized protein derivatized columns than underivatized sorbents. Anionic proteins, in contrast, were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It was concluded that electrostatic adsorption is a prerequisite for analyte protein/immobilized protein interactions of sufficient magnitude to impact ion-exchange separations. Large differences in protein resolution were observed on columns that were identical in all respects except for the immobilized protein, further confirming that analyte/immobilized protein interactions were unique to the interacting pair. The extent of interaction was also influenced by concentration of the immobilized protein in the case of lysozyme. Interactions between the analyte and immobilized protein were found to occur between both the same two proteins and dissimilar species. It was concluded that these phenomena are due to lateral interactions between immobilized proteins and analyte proteins subsequent to electrostatic adsorption of the analyte on the underivatized surface of ion-exchange sorbents.
Ratnayake
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PerSeptive Biosystems Inc., Framingham, MA 01701, USA.
Regnier
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D000180:Acrylic Resins / Q000737:chemistry*; D000327:Adsorption; D000818:Animals; D002411:Cation Exchange Resins / Q000737:chemistry*; D002417:Cattle; D002645:Chickens; D002852:Chromatography, Ion Exchange; D002919:Chymotrypsinogen / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D006736:Horses; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D009994:Osmolar Concentration; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D055672:Static Electricity; D013499:Surface Properties
D000180:Acrylic Resins; D002411:Cation Exchange Resins; D003574:Cytochrome c Group; D004800:Enzymes, Immobilized; D009211:Myoglobin; D011506:Proteins; D012710:Serum Albumin, Bovine; C006903:carbopol 940; D012822:Silicon Dioxide; D002919:Chymotrypsinogen; D009113:Muramidase